Peptides and related molecules that bind to TALL-1

ABSTRACT

The present invention concerns therapeutic agents that modulate the activity of TALL-1. In accordance with the present invention, modulators of TALL-1 may comprise an amino acid sequence Dz 2 Lz 4  wherein z 2  is an amino acid residue and z 4  is threonyl or isoleucyl. Exemplary molecules comprise a sequence of the formulae  
                                     (SEQ. ID. NO: 100)                               a 1 a 2 a 3 CDa 6 La 8 a 9 a 10 Ca 12 a 13 a 14,                                           (SEQ. ID. NO: 104)                               b 1 b 2 b 3 Cb 5 b 6 Db 8 Lb 10 b 11 b 12 b 13 b 14 Cb 16 b 17 b 18                                           (SEQ. ID. NO: 105)                               c 1 c 2 c 3 Cc 5 Dc 7 Lc 9 c 10 c 11 c 12 c 13 c 14 Cc 16 c 17 c 18                                           (SEQ. ID. NO: 106)                               d 1 d 2 d 3 Cd 5 d 6 d 7 WDd 10 Ld 13 d 14 d 15 Cd 16 d 17 d 18                                           (SEQ. ID. NO: 107)                               e 1 e 2 e 3 Ce 5 e 6 e 7 De 9 Le 11 Ke 13 Ce 15 e 16 e 17 e 18                                           (SEQ. ID NO: 109)                               f 1 f 2 f 3 Kf 5 Df 7 Lf 9 f 10 Qf 12 f 13 f 14                                                                                           
 
     wherein the substituents are as defined in the specification. The invention further comprises compositions of matter of the formula 
     (X 1 ) a —V 1 —(X 2 ) b   
     wherein V 1  is a vehicle that is covalently attached to one or more of the above TALL-1 modulating compositions of matter. The vehicle and the TALL-1 modulating composition of matter may be linked through the N- or C-terminus of the TALL-1 modulating portion. The preferred vehicle is an Fc domain, and the preferred Fc domain is an IgG Fc domain.

[0001] This application is related to U.S. provisional application No. 60/290,196, filed May 11, 2001, which is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] After years of study in necrosis of tumors, tumor necrosis factors (TNFs) α and β were finally cloned in 1984. The ensuing years witnessed the emergence of a superfamily of TNF cytokines, including fas ligand (FasL), CD27 ligand (CD27L), CD30 ligand (CD30L), CD40 ligand (CD40L), TNF-related apoptosis-inducing ligand (TRAIL, also designated AGP-1), osteoprotegerin binding protein (OPG-BP or OPG ligand), 4-1BB ligand, LIGHT, APRIL, and TALL-1. Smith et al. (1994), Cell 76: 959-962; Lacey et al. (1998), Cell 93: 165-176; Chichepotiche et al. (1997), J. Biol. Chem. 272: 32401-32410; Mauri et al. (1998), Immunity 8: 21-30; Hahne et al. (1998), J. Exp. Med. 188: 1185-90; Shu et al. (1999), J. Leukocyte Biology 65: 680-3. This family is unified by its structure, particularly at the C-terminus. In addition, most members known to date are expressed in immune compartments, although some members are also expressed in other tissues or organs, as well. Smith et al. (1994), Cell 76: 959-62. All ligand members, with the exception of LT-α, are type II transmembrane proteins, characterized by a conserved 150 amino acid region within C-terminal extracellular domain. Though restricted to only 20-25% identity, the conserved 150 amino acid domain folds into a characteristic β-pleated sheet sandwich and trimerizes. This conserved region can be proteolytically released, thus generating a soluble functional form. Banner et al. (1993), Cell 73: 431-445.

[0003] Many members within this ligand family are expressed in lymphoid enriched tissues and play important roles in the immune system development and modulation. Smith et al. (1994). For example, TNFα is mainly synthesized by macrophages and is an important mediator for inflammatory responses and immune defenses. Tracey & Cerami (1994), Ann. Rev. Med. 45: 491-503. Fas-L, predominantly expressed in activated T cell, modulates TCR-mediated apoptosis of thymocytes. Nagata, S. & Suda, T. (1995) Immunology Today 16: 39-43; Castrim et al. (1996), Immunity 5: 617-27. CD40L, also expressed by activated T cells, provides an essential signal for B cell survival, proliferation and immunoglobulin isotype switching. Noelle (1996), Immunity 4: 415-9.

[0004] The cognate receptors for most of the TNF ligand family members have been identified. These receptors share characteristic multiple cysteine-rich repeats within their extracellular domains, and do not possess catalytic motifs within cytoplasmic regions. Smith et al. (1994). The receptors signal through direct interactions with death domain proteins (e.g. TRADD, FADD, and RIP) or with the TRAF proteins (e.g. TRAF2, TRAF3, TRAF5, and TRAF6), triggering divergent and overlapping signaling pathways, e.g. apoptosis, NF-κB activation, or JNK activation. Wallach et al. (1999), Annual Review of Immunology 17: 331-67. These signaling events lead to cell death, proliferation, activation or differentiation. The expression profile of each receptor member varies. For example, TNFR1 is expressed on a broad spectrum of tissues and cells, whereas the cell surface receptor of OPGL is mainly restricted to the osteoclasts. Hsu et al. (1999) Proc. Natl. Acad. Sci. USA 96: 3540-5.

[0005] A number of research groups have recently identified TNF family ligands with the same or substantially similar sequence. The ligand has been variously named neutrokine α (WO 98/18921, published May 7, 1998),63954 (WO 98/27114, published Jun. 25, 1998), TL5 (EP 869 180, published Oct. 7, 1998), NTN-2 (WO 98/55620 and WO 98/55621, published Dec. 10, 1998), TNRL1-alpha (WO 9911791, published Mar. 11, 1999), kay ligand (WO99/12964, published Mar. 18, 1999), and AGP-3 (U.S. Prov. App. No. 60/119,906, filed Feb. 12, 1999 and No. 60/166,271, filed Nov. 18, 1999, respectively); and TALL-1 (WO 00/68378, published Nov. 16, 2000). Each of these references is hereby incorporated by reference. Hereinafter, the ligands reported therein are collectively referred to as TALL-1.

[0006] TALL-1 is a member of the TNF ligand superfamily that is functionally involved in B cell survival and proliferation. Transgenic mice overexpressing TALL-1 had severe B cell hyperplasia and lupus-like autoimmune disease. Khare et al. (2000) PNAS 97(7):3370-3375). Both TACI and BCMA serve as cell surface receptors for TALL-1. Gross et al. (2000), Nature 404: 995-999; Ware (2000), J. Exp. Med. 192(11): F35-F37; Ware (2000), Nature 404: 949-950; Xia et al. (2000), J. Exp. Med. 192(1):137-143; Yu et al. (2000), Nature Immunology 1(3):252-256; Marsters et al. (2000), Current Biology 10:785-788; Hatzoglou et al. (2000) J. of Immunology 165:1322-1330; Shu et al. (2000) PNAS 97(16):9156-9161; Thompson et al. (2000) J. Exp. Med. 192(1):129-135; Mukhopadhyay et al. (1999) J. Biol. Chem. 274(23): 15978-81; Shu et al. (1999) J. Leukocyte Biol. 65:680-683; Gruss et al. (1995) Blood 85(12): 3378-3404; Smith et al. (1994), Cell 76: 959-962; U.S. Pat. No. 5,969,102, issued Oct. 19, 1999; WO 00/67034, published Nov. 9, 2000; WO 00/40716, published Jul. 13, 2000; WO 99/35170, published Jul. 15, 1999. Both receptors are expressed on B cells and signal through interaction with TRAF proteins. In addition, both TACI and BCMA also bind to another TNF ligand family member, APRIL. Yu et al. (2000), Nature Immunology 1(3) :252-256. APRIL has also been demonstrated to induce B cell proliferation.

[0007] To date, no recombinant or modified proteins employing peptide modulators of TALL-1 have been disclosed. Recombinant and modified proteins are an emerging class of therapeutic agents. Useful modifications of protein therapeutic agents include combination with the “Fc” domain of an antibody and linkage to polymers such as polyethylene glycol (PEG) and dextran. Such modifications are discussed in detail in a patent application entitled, “Modified Peptides as Therapeutic Agents,” publicshed WO 00/24782, which is hereby incorporated by reference in its entirety.

[0008] A much different approach to development of therapeutic agents is peptide library screening. The interaction of a protein ligand with its receptor often takes place at a relatively large interface. However, as demonstrated for human growth hormone and its receptor, only a few key residues at the interface contribute to most of the binding energy. Clackson et al. (1995), Science 267: 383-6. The bulk of the protein ligand merely displays the binding epitopes in the right topology or serves functions unrelated to binding. Thus, molecules of only “peptide” length (2 to 40 amino acids) can bind to the receptor protein of a given large protein ligand. Such peptides may mimic the bioactivity of the large protein ligand (“peptide agonists”) or, through competitive binding, inhibit the bioactivity of the large protein ligand (“peptide antagonists”).

[0009] Phage display peptide libraries have emerged as a powerful method in identifying such peptide agonists and antagonists. See, for example, Scott et al. (1990), Science 249: 386; Devlin et al. (1990), Science 249: 404; U.S. Pat. No. 5,223,409, issued Jun. 29, 1993; U.S. Pat. No. 5,733,731, issued Mar. 31, 1998; U.S. Pat. No. 5,498,530, issued Mar. 12, 1996; U.S. Pat. No. 5,432,018, issued Jul. 11, 1995; U.S. Pat. No. 5,338,665, issued Aug. 16, 1994; U.S. Pat. No. 5,922,545, issued Jul. 13, 1999; WO 96/40987, published Dec. 19, 1996; and WO 98/15833, published Apr. 16, 1998 (each of which is incorporated by reference in its entirety). In such libraries, random peptide sequences are displayed by fusion with coat proteins of filamentous phage. Typically, the displayed peptides are affinity-eluted against an immobilized target protein. The retained phages may be enriched by successive rounds of affinity purification and repropagation. The best binding peptides may be sequenced to identify key residues within one or more structurally related families of peptides. See, e.g., Cwirla et al. (1997), Science 276: 1696-9, in which two distinct families were identified. The peptide sequences may also suggest which residues may be safely replaced by alanine scanning or by mutagenesis at the DNA level. Mutagenesis libraries may be created and screened to further optimize the sequence of the best binders. Lowman (1997), Ann. Rev. Biophys. Biomol. Struct. 26: 401-24.

[0010] Structural analysis of protein-protein interaction may also be used to suggest peptides that mimic the binding activity of large protein ligands. In such an analysis, the crystal structure may suggest the identity and relative orientation of critical residues of the large protein ligand, from which a peptide may be designed. See, e.g., Takasaki et al. (1997), Nature Biotech. 15: 1266-70. These analytical methods may also be used to investigate the interaction between a receptor protein and peptides selected by phage display, which may suggest further modification of the peptides to increase binding affinity.

[0011] Other methods compete with phage display in peptide research. A peptide library can be fused to the carboxyl terminus of the lac repressor and expressed in E. coli. Another E. coli-based method allows display on the cell's outer membrane by fusion with a peptidoglycan-associated lipoprotein (PAL). Hereinafter, these and related methods are collectively referred to as “E. coli display.” In another method, translation of random RNA is halted prior to ribosome release, resulting in a library of polypeptides with their associated RNA still attached. Hereinafter, this and related methods are collectively referred to as “ribosome display.” Other methods employ peptides linked to RNA; for example, PROfusion technology, Phylos, Inc. See, for example, Roberts & Szostak (1997), Proc. Natl. Acad. Sci. USA, 94: 12297-303. Hereinafter, this and related methods are collectively referred to as “RNA-peptide screening.” Chemically derived peptide libraries have been developed in which peptides are immobilized on stable, non-biological materials, such as polyethylene rods or solvent-permeable resins. Another chemically derived peptide library uses photolithography to scan peptides immobilized on glass slides. Hereinafter, these and related methods are collectively referred to as “chemical-peptide screening.” Chemical-peptide screening may be advantageous in that it allows use of D-amino acids and other unnatural analogues, as well as non-peptide elements. Both biological and chemical methods are reviewed in Wells & Lowman (1992), Curr. Opin. Biotechnol. 3: 355-62. Conceptually, one may discover peptide mimetics of any protein using phage display, RNA-peptide screening, and the other methods mentioned above.

SUMMARY OF THE INVENTION

[0012] The present invention concerns therapeutic agents that modulate the activity of TALL-1. In accordance with the present invention, modulators of TALL-1 may comprise an amino acid sequence Dz²Lz⁴ (SEQ ID NO: 108) wherein z² is an amino acid residue and z⁴ is threonyl or isoleucyl. Such modulators of TALL-1 comprise molecules of the following formulae: I(a) a¹a²a³CDa⁶La⁸a⁹a¹⁰Ca¹²a¹³a¹⁴ (SEQ. ID. NO: 100)

[0013] wherein:

[0014] a¹, a², a³ are each independently absent or amino acid residues;

[0015] a⁶ is an amino acid residue;

[0016] a⁹ is a basic or hydrophobic residue;

[0017] a⁸ is threonyl or isoleucyl;

[0018] a¹² is a neutral polar residue; and

[0019] a¹³ and a¹⁴ are each independently absent or amino acid residues. (SEQ. ID. NO: 104) I(b) b¹b²b³Cb⁵b⁶Db³Lb¹⁰b¹¹b¹²b¹³b¹⁴Cb¹⁶b¹⁷b¹⁸

[0020] wherein:

[0021] b¹ and b² are each independently absent or amino acid residues;

[0022] b³ is an acidic or amide residue;

[0023] b⁵ is an amino acid residue;

[0024] b⁶ is an aromatic residue;

[0025] b⁸ is an amino acid residue;

[0026] b¹⁰ T or I;

[0027] b¹¹ is a basic residue;

[0028] b¹² and b¹³ are each independently amino acid residues;

[0029] b¹⁴ is a neutral polar residue; and

[0030] b¹⁶, b¹⁷ and b¹⁸ are each independently absent or amino acid residues. (SEQ.ID. NO: 105) I(c) c¹c²c³Cc⁵Dc⁷Lc⁹c¹⁰c¹¹c¹²c¹³c¹⁴Cc¹⁶c¹⁷c¹⁸

[0031] wherein:

[0032] c¹, c², and c³ are each independently absent or amino acid residues;

[0033] c⁵ is an amino acid residue;

[0034] c⁷ is an amino acid residue;

[0035] c⁹ is T or I;

[0036] c¹⁰ is a basic residue;

[0037] c¹¹ and c¹² are each independently amino acid residues;

[0038] c¹³ is a neutral polar residue;

[0039] c¹⁴ is an amino acid residue;

[0040] c¹⁶ is an amino acid residue;

[0041] c¹⁷ is a neutral polar residue; and

[0042] c¹⁸ is an amino acid residue or is absent. (SEQ. ID. NO: 106) I(d) d¹d²d³Cd⁵d⁶d⁷WDd¹⁰Ld¹²d¹³d¹⁴Cd¹⁵d¹⁶d¹⁷

[0043] wherein:

[0044] d¹, d², and d³ are each independently absent or amino acid residues;

[0045] d⁵, d⁶, and d⁷ are each independently amino acid residues;

[0046] d¹⁰ is an amino acid residue;

[0047] d¹³ is T or I;

[0048] d¹⁴ is an amino acid residue; and

[0049] d¹⁶, d¹⁷, and d¹⁸ are each independently absent or amino acid residues. (SEQ. ID. NO: 107) I(e) e¹e²e³Ce⁵e⁶e⁷De⁹Le¹¹Ke¹³Ce¹⁵e¹⁶e¹⁷e¹⁸

[0050] wherein:

[0051] e¹, e², and e³ are each independently absent or amino acid residues;

[0052] e⁵, e⁶, e⁷, e⁹, and e¹³ are each independently amino acid residues;

[0053] e¹¹ is T or I; and

[0054] e¹⁵, e¹⁶, and e¹⁷ are each independently absent or amino acid residues. I(f) f¹f²f³Kf⁵Df⁷Lf⁹f¹⁰Qf¹²f¹³f¹⁴ (SEQ. ID NO: 109)

[0055] wherein:

[0056] f¹, f², and f³ are absent or are amino acid residues (with one of f¹, f², and f³ preferred to be C when one of f¹², f¹³, and f¹⁴ is C);

[0057] f⁵ is W, Y, or F (W preferred);

[0058] f⁷ is an amino acid residue (L preferred);

[0059] f⁹ is T or I (T preferred);

[0060] f¹⁰ is K, R, or H (K preferred);

[0061] f¹² is C, a neutral polar residue, or a basic residue (W, C, or R preferred);

[0062] f¹³ is C, a neutral polar residue or is absent (V preferred); and

[0063] f¹⁴ is any amino acid residue or is absent;

[0064] provided that only one of f¹, f², and f³ may be C, and only one of f¹², f¹³, and f¹⁴ may be C.

[0065] Compounds of formulae I(a) through I(f) above incorporate Dz²Lz⁴, as well as SEQ ID NO: 63 hereinafter. The sequence of I(f) was derived as a consensus sequence as described in Example 1 hereinbelow. Of compounds within formula I(f), those within the formula I(f′) f¹f²f³KWDf⁷Lf⁹KQf¹²f¹³f¹⁴ (SEQ ID NO: 125)

[0066] are preferred. Compounds falling within formula I(f′) include SEQ ID NOS: 32, 58, 60, 62, 63, 66, 67, 69, 70, 114, 115, 122, 123, 124, 147-150, 152-177, 179, 180, 187.

[0067] Also in accordance with the present invention are compounds having the consensus motif: PFPWE (SEQ ID NO: 110)

[0068] which also bind TALL-1.

[0069] Further in accordance with the present invention are compounds of the formulae: I(g) g¹g²g³Cg⁵PFg⁸Wg¹⁰Cg¹¹g¹²g¹³ (SEQ. ID. NO. 101)

[0070] wherein:

[0071] g¹, g² and g³ are each independently absent or amino acid residues;

[0072] g⁵ is a neutral polar residue;

[0073] g⁸ is a neutral polar residue;

[0074] g¹⁰ is an acidic residue;

[0075] g¹² and g¹³ are each independently amino acid residues; and

[0076] g¹⁴ is absent or is an amino acid residue. I(h) h¹h²h³CWh⁶h⁷WGh¹⁰Ch¹²h¹³h¹⁴ (SEQ. ID. NO: 102)

[0077] wherein:

[0078] h¹, h², and h³ are each independently absent or amino acid residues;

[0079] h⁶ is a hydrophobic residue;

[0080] h⁷ is a hydrophobic residue;

[0081] h¹⁰ is an acidic or polar hydrophobic residue; and

[0082] h¹², h¹³, and h¹⁴ are each independently absent or amino acid residues. I(i) i¹i²i³Ci⁵i⁶i⁷i⁸i⁹i¹⁰Ci¹²i¹³i¹⁴ (SEQ. ID. NO: 103)

[0083] wherein:

[0084] i¹ is absent or is an amino acid residue;

[0085] i² is a neutral polar residue;

[0086] i³ is an amino acid residue;

[0087] i⁵, i⁶, i⁷, and i⁸ are each independently amino acid residues;

[0088] i⁹ is an acidic residue;

[0089] i¹⁰ is an amino acid residue;

[0090] i¹² and i¹³ are each independently amino acid residues; and

[0091] i¹⁴ is a neutral polar residue.

[0092] The compounds defined by formulae I(g) through I(i) also bind TALL-1.

[0093] Further in accordance with the present invention, modulators of TALL-1 comprise:

[0094] a) a TALL-1 modulating domain (e.g., an amino acid sequence of Formulae I(a) through I(i)), preferably the amino acid sequence Dz²Lz⁴, or sequences derived therefrom by phage display, RNA-peptide screening, or the other techniques mentioned above; and

[0095] b) a vehicle, such as a polymer (e.g., PEG or dextran) or an Fc domain, which is preferred;

[0096] wherein the vehicle is covalently attached to the TALL-1 modulating domain. The vehicle and the TALL-1 modulating domain may be linked through the N- or C-terminus of the TALL-1 modulating domain, as described further below. The preferred vehicle is an Fc domain, and the preferred Fc domain is an IgG Fc domain. Such Fc-linked peptides are referred to herein as “peptibodies.” Preferred TALL-1 modulating domains comprise the amino acid sequences described hereinafter in Tables 1 and 2. Other TALL-1 modulating domains can be generated by phage display, RNA-peptide screening and the other techniques mentioned herein.

[0097] Further in accordance with the present invention is a process for making TALL-1 modulators, which comprises:

[0098] a. selecting at least one peptide that binds to TALL-1; and

[0099] b. covalently linking said peptide to a vehicle. The preferred vehicle is an Fc domain. Step (a) is preferably carried out by selection from the peptide sequences in Table 2 hereinafter or from phage display, RNA-peptide screening, or the other techniques mentioned herein.

[0100] The compounds of this invention may be prepared by standard synthetic methods, recombinant DNA techniques, or any other methods of preparing peptides and fusion proteins. Compounds of this invention that encompass non-peptide portions may be synthesized by standard organic chemistry reactions, in addition to standard peptide chemistry reactions when applicable.

[0101] The primary use contemplated for the compounds of this invention is as therapeutic or prophylactic agents. The vehicle-linked peptide may have activity comparable to—or even greater than—the natural ligand mimicked by the peptide.

[0102] The compounds of this invention may be used for therapeutic or prophylactic purposes by formulating them with appropriate pharmaceutical carrier materials and administering an effective amount to a patient, such as a human (or other mammal) in need thereof. Other related aspects are also included in the instant invention.

[0103] Numerous additional aspects and advantages of the present invention will become apparent upon consideration of the figures and detailed description of the invention.

BRIEF DESCRIPTION OF THE FIGURES

[0104]FIG. 1 shows exemplary Fc dimers that may be derived from an IgG1 antibody. “Fc” in the figure represents any of the Fc variants within the meaning of “Fc domain” herein. “X¹” and “X²” represent peptides or linker-peptide combinations as defined hereinafter. The specific dimers are as follows:

[0105] A, D: Single disulfide-bonded dimers. IgG1 antibodies typically have two disulfide bonds at the hinge region of the antibody. The Fc domain in FIGS. 1A and 1D may be formed by truncation between the two disulfide bond sites or by substitution of a cysteinyl residue with an unreactive residue (e.g., alanyl). In FIG. 1A, the Fc domain is linked at the amino terminus of the peptides; in 1D, at the carboxyl terminus.

[0106] B, E: Doubly disulfide-bonded dimers. This Fc domain may be formed by truncation of the parent antibody to retain both cysteinyl residues in the Fc domain chains or by expression from a construct including a sequence encoding such an Fc domain. In FIG. 1B, the Fc domain is linked at the amino terminus of the peptides; in 1E, at the carboxyl terminus.

[0107] C, F: Noncovalent dimers. This Fc domain may be formed by elimination of the cysteinyl residues by either truncation or substitution. One may desire to eliminate the cysteinyl residues to avoid impurities formed by reaction of the cysteinyl residue with cysteinyl residues of other proteins present in the host cell. The noncovalent bonding of the Fc domains is sufficient to hold together the dimer. Other dimers may be formed by using Fc domains derived from different types of antibodies (e.g., IgG2, IgM).

[0108]FIG. 2 shows the structure of preferred compounds of the invention that feature tandem repeats of the pharmacologically active peptide. FIG. 2A shows a single chain molecule and may also represent the DNA construct for the molecule. FIG. 2B shows a dimer in which the linker-peptide portion is present on only one chain of the dimer. FIG. 2C shows a dimer having the peptide portion on both chains. The dimer of FIG. 2C will form spontaneously in certain host cells upon expression of a DNA construct encoding the single chain shown in FIG. 3A. In other host cells, the cells could be placed in conditions favoring formation of dimers or the dimers can be formed in vitro.

[0109]FIG. 3 shows exemplary nucleic acid and amino acid sequences (SEQ ID NOS: 1 and 2, respectively) of human IgG1 Fc that may be used in this invention.

[0110]FIGS. 4A through 4F show the nucleotide and amino acid sequences (SEQ ID NOS: 3-27) S of NdeI to SalI fragments encoding peptide and linker.

[0111]FIGS. 5A through 5M show the nucleotide sequence (SEQ ID NO: 28) of pAMG21-RANK-Fc vector, which was used to construct Fc-linked molecules of the present invention. These figures identify a number of features of the nucleic acid, including:

[0112] promoter regions PcopB, PrepA, RNAI, APHII, luxPR, and luxPL;

[0113] mRNA for APHII, luxR;

[0114] coding sequences and amino acid sequences for the proteins copB protein, copT, repAI, repA4, APHII, luxR, RANK, and Fc;

[0115] binding sites for the proteins copB, CRP;

[0116] hairpins T1, T2, T7, and toop;

[0117] operator site for lux protein;

[0118] enzyme restriction sites for Pfll108I, BglII, ScaI, BmnI, DrdII, DraIII, BstBI, AceIII, AflII, PflMI, BglI, SfiI, BstEII, BspLullI, NspV, BplI, EagI, BcgI, NsiI, BsaI, Psp1406I, AatII, BsmI, NruI, NdeI, ApaLI, Acc65I, KpnI, SalI, AccI, BspEI, AhdI, BspHI, EconI, BsrGI, BmaI, SmaI, SexAI, BamHI, and BlpI.

[0119]FIGS. 6A and 6B show the DNA sequence (SEQ ID NO: 97) inserted into pCFM1656 between the unique AatII (position #4364 in pCFM1656) and SacII (position #4585 in pCFM1656) restriction sites to form expression plasmid pAMG21 (ATCC accession no. 98113).

[0120]FIG. 7 shows that the TALL-1 peptibody (SEQ ID NO: 70) inhibits TALL-1 mediated B cell proliferation. Purified B cells (10 ⁵) from B6 mice were cultured in triplicates in 96-well plated with the indicated amounts of TALL-1 consensus peptibody in the presence of 10 ng/ml TALL-1 plus 2 μg/ml anti-IgM antibody. Proliferation was measured by radioactive [³H]thymidine uptake in the last 18 h of pulse. Data shown represent mean±SD triplicate wells.

[0121]FIG. 8 shows that a TALL-1 N-terminal tandem dimer peptibodies (SEQ ID NO: 123, 124 in Table 5B hereinafter) are preferable for inhibition of TALL-1-mediated B cell proliferation. Purified B cells (10 ⁵) from B6 mice were cultured in triplicates in 96-well plated with the indicated amounts of TALL-1 12-3 peptibody and TALL-1 consensus peptibody (SEQ ID NOS: 115 and 122 of Table 5B)or the related dimer peptibodies (SEQ ID NOS: 123, 124) in the presence of 10 ng/ml TALL-1 plus 2 μg/ml anti-IgM antibody. Proliferation was measured by radioactive [³H]thymidine uptake in the last 18 h of pulse. Data shown represent mean±SD triplicate wells.

[0122]FIG. 9. AGP3 peptibody binds to AGP3 with high affinity. Dissociation equilibrium constant (K_(D)) was obtained from nonlinear regression of the competition curves using a dual-curve one-site homogeneous binding model (KinEx™ software). K_(D) is about 4 pM for AGP3 peptibody binding with human AGP3 (SEQ ID NO: 123).

[0123]FIGS. 10A and 10B. AGP3 peptibody blocks both human and murine AGP3 in the Biacore competition assay. Soluble human TACI protein was immobilized to B1 chip. 1 nM of recombinant human AGP3 protein (upper panel) or 5 nM of recombinant murine AGP3 protein (lower panel) was incubated with indicated amount of AGP3 peptibody before injected over the surface of receptor. Relative human AGP3 and murine AGP3 (binding response was shown (SEQ ID NO: 123).

[0124]FIGS. 11A and 11B. AGP3 peptibody blocked AGP3 binding to all three receptors TACI, BCMA and BAFFR in Biacore competition assay. Recombinant soluble receptor TACI, BCMA and BAFFR proteins were immobilized to CM5 chip. 1 nM of recombinant human AGP3 (upper panel) were incubated with indicated amount of AGP3 peptibody before injected over each receptor surface. Relative binding of AGP3 was measured. Similarly, 1 nM of recombinant APRIL protein was incubated with indicated amount of AGP3 peptibody before injected over each receptor surface. AGP3 peptibody didn't inhibit APRIL binding to all three receptors (SEQ ID NO: 123).

[0125]FIGS. 12A and 12B. AGP3 peptibody inhibits mouse serum immunoglobulin level increase induced by human AGP3 challenge. Balb/c mice received 7 daily intraperitoneal injections of 1 mg/Kg human AGP3 protein along with saline, human Fc, or AGP3 peptibody at indicated doses, and were bled on day 8. Serum total IgM and IgA level were measured by ELISA (SEQ ID NO: 123).

[0126]FIG. 13. AGP3 peptibody treatment reduced arthritis severity in the mouse CIA model. Eight to 12 weeks old DBA/1 male mice were immunized with bovine collagen type II (bCII) emulsified in complete freunds adjuvant intradermally at the base of tail, and were boosted 3 weeks after the initial immunization with bCII emulsified in incomplete freunds adjuvant. Treatment with indicated dosage of AGP3 peptibody was begun from the day of booster immunization for 4 weeks. As described before (Khare et al., J. Immunol., 155: 3653-9, 1995), all four paws were individually scored from 0-3 for arthritis severity (SEQ ID NO: 123).

[0127]FIG. 14. AGP3 peptibody treatment inhibited anti-collagen antibody generation in the mouse CIA model. Serum samples were taken one week after final treatment (day 35) as described above. Serum anti-collagen II antibody level was determined by ELISA analysis (SEQ ID NO: 123).

[0128]FIGS. 15A and 15B. AGP3 peptibody treatment delayed proteinuria onset and improved survival in NZB/NZW lupus mice. Five-month-old lupus prone NZBx NZBWF1 mice were treated i.p. 3×/week for 8 weeks with PBS or indicated doses of AGP3 peptibody (SEQ ID NO: 123) or human Fc proteins. Protein in the urine was evaluated monthly throughout the life of the experiment with Albustix reagent strips (Bayer AG).

[0129]FIGS. 16A and 16B show the nucleic acid and amino acid sequences of a preferred TALL-1-binding peptibody (SEQ ID NOS: 189 and 123)

DETAILED DESCRIPTION OF THE INVENTION

[0130] Definition of Terms

[0131] The terms used throughout this specification are defined as follows, unless otherwise limited in specific instances.

[0132] General Definitions

[0133] The term “comprising” means that a compound may include additional amino acids on either or both of the N- or C-termini of the given sequence. Of course, these additional amino acids should not significantly interfere with the activity of the compound.

[0134] Additionally, physiologically acceptable salts of the compounds of this invention are also encompassed herein. The term “physiologically acceptable salts” refers to any salts that are known or later discovered to be pharmaceutically acceptable. Some specific examples are: acetate; trifluoroacetate; hydrohalides, such as hydrochloride and hydrobromide; sulfate; citrate; tartrate; glycolate; and oxalate.

[0135] Amino Acids

[0136] The term “acidic residue” refers to amino acid residues in D- or L-form having sidechains comprising acidic groups. Exemplary acidic residues include D and E.

[0137] The term “amide residue” refers to amino acids in D- or L-form having sidechains comprising amide derivatives of acidic groups. Exemplary residues include N and Q.

[0138] The term “aromatic residue” refers to amino acid residues in D- or L-form having sidechains comprising aromatic groups. Exemplary aromatic residues include F, Y, and W.

[0139] The term “basic residue” refers to amino acid residues in D- or L-form having sidechains comprising basic groups. Exemplary basic residues include H, K, and R.

[0140] The term “hydrophilic residue” refers to amino acid residues in D- or L-form having sidechains comprising polar groups. Exemplary hydrophilic residues include C, S, T, N, and Q.

[0141] The term “nonfunctional residue ” refers to amino acid residues in D- or L-form having sidechains that lack acidic, basic, or aromatic groups. Exemplary nonfunctional amino acid residues include M, G, A, V, I, L and norleucine (Nle).

[0142] The term “neutral polar residue” refers to amino acid residues in D- or L-form having sidechains that lack basic, acidic, or polar groups. Exemplary neutral polar amino acid residues include A, V, L, I, P, W, M, and F.

[0143] The term “polar hydrophobic residue” refers to amino acid residues in D- or L-form having sidechains comprising polar groups. Exemplary polar hydrophobic amino acid residues include T, G, S, Y, C, Q, and N.

[0144] The term “hydrophobic residue” refers to amino acid residues in D- or L-form having sidechains that lack basic or acidic groups. Exemplary hydrophobic amino acid residues include A, V, L, I, P, W, M, F, T, G, S, Y, C, Q, and N.

[0145] Peptides

[0146] The term “peptide” refers to molecules of 1 to 40 amino acids, with molecules of 5 to 20amino acids preferred. Exemplary peptides may comprise the TALL-1 modulating domain of a naturally occurring molecule or comprise randomized sequences.

[0147] The term “randomized” as used to refer to peptide sequences refers to fully random sequences (e.g., selected by phage display methods or RNA-peptide screening) and sequences in which one or more residues of a naturally occurring molecule is replaced by an amino acid residue not appearing in that position in the naturally occurring molecule. Exemplary methods for identifying peptide sequences include phage display, E. coli display, ribosome display, RNA-peptide screening, chemical screening, and the like.

[0148] The term “TALL-1 modulating domain” refers to any amino acid sequence that binds to the TALL-1 and comprises naturally occurring sequences or randomized sequences. Exemplary TALL-1 modulating domains can be identified or derived by phage display or other methods mentioned herein.

[0149] The term “TALL-1 antagonist” refers to a molecule that binds to the TALL-1 and increases or decreases one or more assay parameters opposite from the effect on those parameters by full length native TALL-1. Such activity can be determined, for example, by such assays as described in the subsection entitled “Biological activity of AGP-3” in the Materials & Methods section of the patent application entitled, “TNF-RELATED PROTEINS”, WO 00/47740, published Aug. 17, 2000.

[0150] Vehicles and Peptibodies

[0151] The term “vehicle” refers to a molecule that prevents degradation and/or increases half-life, reduces toxicity, reduces immunogenicity, or increases biological activity of a therapeutic protein. Exemplary vehicles include an Fc domain (which is preferred) as well as a linear polymer (e.g., polyethylene glycol (PEG), polylysine, dextran, etc.); a branched-chain polymer (see, for example, U.S. Pat. No. 4,289,872 to Denkenwalter et al., issued Sep. 15, 1981; 5,229,490 to Tam, issued Jul. 20, 1993; WO 93/21259 by Frechet et al., published Oct. 28, 1993); a lipid; a cholesterol group (such as a steroid); a carbohydrate or oligosaccharide (e.g., dextran); any natural or synthetic protein, polypeptide or peptide that binds to a salvage receptor; albumin, including human serum albumin (HSA), leucine zipper domain, and other such proteins and protein fragments. Vehicles are further described hereinafter.

[0152] The term “native Fc” refers to molecule or sequence comprising the sequence of a non-antigen-binding fragment resulting from digestion of whole antibody, whether in monomeric or multimeric form. The original immunoglobulin source of the native Fc is preferably of human origin and may be any of the immunoglobulins, although IgG1 and IgG2 are preferred. Native Fc's are made up of monomeric polypeptides that may be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent association. The number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4 depending on class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2). One example of a native Fc is a disulfide-bonded dimer resulting from papain digestion of an IgG (see Ellison et al. (1982), Nucleic Acids Res. 10: 4071-9). The term “native Fc” as used herein is generic to the monomeric, dimeric, and multimeric forms.

[0153] The term “Fc variant” refers to a molecule or sequence that is modified from a native Fc but still comprises a binding site for the salvage receptor, FcRn. International applications WO 97/34631 (published Sep. 25, 1997) and WO 96/32478 describe exemplary Fc variants, as well as interaction with the salvage receptor, and are hereby incorporated by reference in their entirety. Thus, the term “Fc variant” comprises a molecule or sequence that is humanized from a non-human native Fc. Furthermore, a native Fc comprises sites that may be removed because they provide structural features or biological activity that are not required for the fusion molecules of the present invention. Thus, the term “Fc variant” comprises a molecule or sequence that lacks one or more native Fc sites or residues that affect or are involved in (1) disulfide bond formation, (2) incompatibility with a selected host cell (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to an Fc receptor other than a salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC). Fc variants are described in further detail hereinafter.

[0154] The term “Fc domain” encompasses native Fc and Fc variant molecules and sequences as defined above. As with Fc variants and native Fc's, the term “Fc domain” includes molecules in monomeric or multimeric form, whether digested from whole antibody or produced by other means.

[0155] The term “multimer” as applied to Fc domains or molecules comprising Fc domains refers to molecules having two or more polypeptide chains associated covalently, noncovalently, or by both covalent and non-covalent interactions. IgG molecules typically form dimers; IgM, pentamers; IgD, dimers; and IgA, monomers, dimers, trimers, or tetramers. Multimers may be formed by exploiting the sequence and resulting activity of the native Ig source of the Fc or by derivatizing (as defined below) such a native Fc.

[0156] The term “dimer” as applied to Fc domains or molecules comprising Fc domains refers to molecules having two polypeptide chains associated covalently or non-covalently. Thus, exemplary dimers within the scope of this invention are as shown in FIG. 1.

[0157] The terms “derivatizing” and “derivative” or “derivatized” comprise processes and resulting compounds respectively in which (1) the compound has a cyclic portion; for example, cross-linking between cysteinyl residues within the compound; (2) the compound is cross-linked or has a cross-linking site; for example, the compound has a cysteinyl residue and thus forms cross-linked dimers in culture or in vivo; (3) one or more peptidyl linkage is replaced by a non-peptidyl linkage; (4) the N-terminus is replaced by —NRR¹, NRC(O)R¹, —NRC(O)OR¹, —NRS(O)₂R¹, —NHC(O)NHR, a succinimide group, or substituted or unsubstituted benzyloxycarbonyl-NH—, wherein R and R¹ and the ring substituents are as defined hereinafter; (5) the C-terminus is replaced by —C(O)R² or —NR³R⁴ wherein R², R³ and R⁴ are as defined hereinafter; and (6) compounds in which individual amino acid moieties are modified through treatment with agents capable of reacting with selected side chains or terminal residues. Derivatives are further described hereinafter.

[0158] The terms “peptibody” and “peptibodies” refer to molecules comprising an Fc domain and at least one peptide. Such peptibodies may be multimers or dimers or fragments thereof, and they may be derivatized. In the present invention, the molecules of formulae II through VI hereinafter are peptibodies when V¹ is an Fc domain.

[0159] Structure of Compounds

[0160] In General. The present inventors identified sequences capable of binding to and modulating the biological activity of TALL-1. These sequences can be modified through the techniques mentioned above by which one or more amino acids may be changed while maintaining or even improving the binding affinity of the peptide.

[0161] In the compositions of matter prepared in accordance with this invention, the peptide(s) may be attached to the vehicle through the peptide's N-terminus or C-terminus. Any of these peptides may be linked in tandem (i.e., sequentially), with or without linkers. Thus, the vehicle-peptide molecules of this invention may be described by the following formula:

(X¹)_(a)—V¹—(X²)_(b)  II

[0162] wherein:

[0163] V¹ is a vehicle (preferably an Fc domain);

[0164] X¹ and X² are each independently selected from —(L¹)_(c)—P¹,—(L¹)_(c)—P¹—(L²)_(d)—P²,—(L¹)_(c)—P¹—(L²)_(d)—P²—(L³)_(e)—P³, and —(L¹)_(c)—P¹—(L²)_(d)—P²—(L³)_(e)—P³—(L⁴)_(f)—P⁴

[0165] P¹, P², P³, and P⁴ are each independently sequences of TALL-1 modulating domains, such as those of Formulae I(a) through I(i);

[0166] L¹, L², L³, and L⁴ are each independently linkers; and

[0167] a, b, c, d, e, and f are each independently 0 or 1, provided that at least one of a and b is 1.

[0168] Thus, compound II comprises preferred compounds of the formulae

X¹—V¹  III

[0169] and multimers thereof wherein V¹ is an Fc domain and is attached at the C-terminus of A¹;

V¹—X²  IV

[0170] and multimers thereof wherein V¹ is an Fc domain and is attached at the N-terminus of A²;

V¹—(L¹)_(c)—P¹  V

[0171] and multimers thereof wherein V¹ is an Fc domain and is attached at the N-terminus of —(L¹)_(c)—P¹; and

V¹—(L¹)_(c)—P¹—(L²)_(d)—P²  VI

[0172] and multimers thereof wherein V¹ is an Fc domain and is attached at the N-terminus of —L¹—P¹—L²—P².

[0173] Peptides. The peptides of this invention are useful as TALL-1 modulating peptides or as TALL-1 modulating domains in the molecules of formulae II through VI. Molecules of this invention comprising these peptide sequences may be prepared by methods known in the art.

[0174] Preferred peptide sequences are those of the foregoing formulae I(a) having the substituents identified below. TABLE 1 Preferred peptide substituents Formula I(a) a⁸ is T; a⁹ is a basic residue (K most preferred); and a¹² is a neutral polar residue (F most preferred). Formula I(b) b³ is D, Q, or E; b⁶ is W or Y; b¹⁰ is T; b¹¹ is K or R; and b¹⁴ is V or L. Formula I(c) c⁹ is T; c¹⁰ is K or R; c¹³ is a I, L, or V; and c¹⁷ is A or L. Formula I(d) d¹³ is T. Formula I(e) e¹¹ is T. Formula I(f) f⁶ is T; f⁷ is K; and f¹⁰ is V. Formula I(g) g⁵ is W; g⁸ is P; g¹⁰ is E; and g¹³ is a basic residue. Formula I(h) h¹ is G; h⁶ is A; h⁷ is a neutral polar residue; and h¹⁰ is an acidic residue. Formula I(i) i² is W; and i¹⁴ is W.

[0175] Preferred peptide sequences appear in Table 2 below. TABLE 2 Preferred TALL-1 modulating domains SEQ Sequence ID NO: PGTCFPFPWECTHA 29 WGACWPFPWECFKE 30 VPFCDLLTKHCFEA 31 GSRCKYKWDVLTKQCFHH 32 LPGCKWDLLIKQWVCDPL 33 SADCYFDILTKSDVCTSS 34 SDDCMYDQLTRMFICSNL 35 DLNCKYDELTYKEWCQFN 36 FHDCKYDLLTRQMVCHGL 37 RNHCFWDHLLKQDICPSP 38 ANQCWWDSLTKKNVCEFF 39 YKGRQMWDILTRSWVVSL 126 QDVGLWWDILTRAWMPNI 127 QNAQRVWDLLIRTWVYPQ 128 GWNEAWWDELTKIWVLEQ 129 RITCDTWDSLIKKCVPQS 130 GAIMQFWDSLTKTWLRQS 131 WLHSGWWDPLTKHWLQKV 132 SEWFFWFDPLTRAQLKFR 133 GVWFWWFDPLTKQWTQAG 134 MQCKGYYDILTKWCVTNG 135 LWSKEVWDILTKSWVSQA 136 KAAGWWFDWLTKVWVPAP 137 AYQTWFWDSLTRLWLSTT 138 SGQHFWWDLLTRSWTPST 139 LGVGQKWDPLTKQWVSRC 140 VGKMCQWDPLIKRTVCVG 141 CRQGAKFDLLTKQCLLGR 142 GQAIRHWDVLTKQWVDSQ 143 RGPCGSWDLLTKHCLDSQ 144 WQWKQQWDLLTKQMVWVG 145 PITICRKDLLTKQVVCLD 146 KTCNGKWDLLTKQCLQQA 147 KCLKGKWDLLTKQCVTEV 148 RCWNGKWDLLTKQCIHPW 149 NRDMRKWDPLIKQWIVRP 150 QAAAATWDLLTKQWLVPP 151 PEGGPKWDPLTKQFLPPV 152 QTPQKKWDLLTKQWFTRN 153 IGSPCKWDLLTKQMICQT 154 CTAAGKWDLLTKQCIQEK 155 VSQCMKWDLLTKQCLQGW 156 VWGTWKWDLLTKQYLPPQ 157 GWWEMKWDLLTKQWYRPQ 158 TAQVSKWDLLTKQWLPLA 159 QLWGTKWDLLTKQWVQNM 160 WATSQKWDLLTKQWVQNM 161 QRQCAKWDLLTKQCVLFY 162 KTTDCKWDLLTKQRICQV 163 LLCQCKWDLLTKQCLKLR 164 LMWFWKWDLLTKQLVPTF 165 QTWAWKWDLLTKQWIGPM 166 NKELLKWDLLTKQCRGRS 167 GQKDLKWDLLTKQYVRQS 168 PKPCQKWDLLTKQCLGSV 169 GQTGWKWDLLTKQWIQTR 170 VWLDWKWDLLTKQWIHPQ 171 QEWEYKWDLLTKQWGWLR 172 HWDSWKWDLLTKQWVVQA 173 TRPLQKWDLLTKQWLRVG 174 SDQWQKWDLLTKQWFWDV 175 QQTFMKWDLLTKQWTRRH 176 QGECRKWDLLTKQCFPGQ 177 CQMGWRWDPLIKMCLGPS 178 QLDGCKWDLLTKQKVCIP 179 HGYWQKWDLLTKQWVSSE 180 HQGQCGWDLLTRIYLPCH 181 LHKACKWDLLTKQCWPMQ 182 CPPGSVWDLLTKIWIQTC 183 ITQDWRFDTLTRLWLPLR 184 QGGFAAWDVLTKMWITVP 185 GHGTPWWDALTRIWTLGV 186 VWPWQKWDLLTKQFVFQD 187 WQWSWKWDLLTRQYISSS 188 NQTLWKWDLLTKQFITYM 60 PVYQGWWDTLTKLYIWDG 61 WLDGGWRDPLIKRSVQLG 62 GHQQFKWDLLTKQWVQSN 63 QRVGQFWDBLTKMFITGS 64 QAQGWSYDALIKTWIRWP 65 GWMHWKWDPLTKQALPWM 66 GHPTYKWDLLTKQWILQM 67 WNNWSLWDPLTKLWLQQN 68 WQWGWKWDLLTKQWVQQQ 69 GQMCWRWDPLTKMWLGTS 70

[0176] It is noted that the known receptors for TALL-1 bear some sequence homology with preferred peptides: 12-3                      LPGCKWDLLIKQWVCDPL BAFFR   MRRGPRSLRGRDAPVPTPCVPTECYDLLVRKCVDCRLL TACI TICNHQSQRTCAAFCRSLSCRKEQGKFYDHLLRDCISCASI BCMA FVSPSQEIRGRFRRMLQMAGQCSQNEYFDSLLHACIPCQLRC (SEQ ID NOS: 33, 195, 196, and 197, respectively).

[0177] Any peptide containing a cysteinyl residue may be cross-linked with another Cys-containing peptide, either or both of which may be linked to a vehicle. Any peptide having more than one Cys residue may form an intrapeptide disulfide bond, as well. Any of these peptides may be derivatized as described hereinafter.

[0178] Additional useful peptide sequences may result from conservative and/or non-conservative modifications of the amino acid sequences of the sequences in Table 2.

[0179] Conservative modifications will produce peptides having functional and chemical characteristics similar to those of the peptide from which such modifications are made. In contrast, substantial modifications in the functional and/or chemical characteristics of the peptides may be accomplished by selecting substitutions in the amino acid sequence that differ significantly in their effect on maintaining (a) the structure of the molecular backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the size of the molecule.

[0180] For example, a “conservative amino acid substitution” may involve a substitution of a native amino acid residue with a nonnative residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position. Furthermore, any native residue in the polypeptide may also be substituted with alanine, as has been previously described for “alanine scanning mutagenesis” (see, for example, MacLennan et al., 1998, Acta Physiol. Scand. Suppl. 643:55-67; Sasaki et al., 1998, Adv. Biophys. 35:1-24, which discuss alanine scanning mutagenesis).

[0181] Desired amino acid substitutions (whether conservative or non-conservative) can be determined by those skilled in the art at the time such substitutions are desired. For example, amino acid substitutions can be used to identify important residues of the peptide sequence, or to increase or decrease the affinity of the peptide or vehicle-peptide molecules (see preceding formulae) described herein. Exemplary amino acid substitutions are set forth in Table 3. TABLE 3 Amino Acid Substitutions Original Exemplary Preferred Residues Substitutions Substitutions Ala (A) Val, Leu, Ile Val Arg (R) Lys, Gln, Asn Lys Asn (N) Gln Gln Asp (D) Glu Glu Cys (C) Ser, Ala Ser Gln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro, Ala Ala His (H) Asn, Gln, Lys, Arg Arg Ile (I) Leu, Val, Met, Ala, Leu Phe, Norleucine Leu (L) Norleucine, Ile, Val, Ile Met, Ala, Phe Lys (K) Arg, 1,4 Diamino- Arg butyric Acid, Gln, Asn Met (M) Leu, Phe, Ile Leu Phe (F) Leu, Val, Ile, Ala, Tyr Leu Pro (P) Ala Gly Ser (S) Thr, Ala, Cys Thr Thr (T) Ser Ser Trp (W) Tyr, Phe Tyr Tyr (Y) Trp, Phe, Thr, Ser Phe Val (V) Ile, Met, Leu, Phe, Leu Ala, Norleucine

[0182] In certain embodiments, conservative amino acid substitutions also encompass non-naturally occurring amino acid residues which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems.

[0183] As noted in the foregoing section “Definition of Terms,” naturally occurring residues may be divided into classes based on common sidechain properties that may be useful for modifications of sequence. For example, non-conservative substitutions may involve the exchange of a member of one of these classes for a member from another class. Such substituted residues may be introduced into regions of the peptide that are homologous with non-human orthologs, or into the non-homologous regions of the molecule. In addition, one may also make modifications using P or G for the purpose of influencing chain orientation.

[0184] In making such modifications, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

[0185] The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is understood in the art. Kyte et al., J. Mol. Biol., 157:105-131 (1982). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0186] It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. The greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e. with a biological property of the protein.

[0187] The following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). In making changes based upon similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. One may also identify epitopes from primary amino acid sequences on the basis of hydrophilicity. These regions are also referred to as “epitopic core regions.”

[0188] A skilled artisan will be able to determine suitable variants of the polypeptide as set forth in the foregoing sequences using well known techniques. For identifying suitable areas of the molecule that may be changed without destroying activity, one skilled in the art may target areas not believed to be important for activity. For example, when similar polypeptides with similar activities from the same species or from other species are known, one skilled in the art may compare the amino acid sequence of a peptide to similar peptides. With such a comparison, one can identify residues and portions of the molecules that are conserved among similar polypeptides. It will be appreciated that changes in areas of a peptide that are not conserved relative to such similar peptides would be less likely to adversely affect the biological activity and/or structure of the peptide. One skilled in the art would also know that, even in relatively conserved regions, one may substitute chemically similar amino acids for the naturally occurring residues while retaining activity (conservative amino acid residue substitutions). Therefore, even areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the peptide structure.

[0189] Additionally, one skilled in the art can review structure-function studies identifying residues in similar peptides that are important for activity or structure. In view of such a comparison, one can predict the importance of amino acid residues in a peptide that correspond to amino acid residues that are important for activity or structure in similar peptides. One skilled in the art may opt for chemically similar amino acid substitutions for such predicted important amino acid residues of the peptides.

[0190] One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar polypeptides. In view of that information, one skilled in the art may predict the alignment of amino acid residues of a peptide with respect to its three dimensional structure. One skilled in the art may choose not to make radical changes to amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules. Moreover, one skilled in the art may generate test variants containing a single amino acid substitution at each desired amino acid residue. The variants can then be screened using activity assays know to those skilled in the art. Such data could be used to gather information about suitable variants. For example, if one discovered that a change to a particular amino acid residue resulted in destroyed, undesirably reduced, or unsuitable activity, variants with such a change would be avoided. In other words, based on information gathered from such routine experiments, one skilled in the art can readily determine the amino acids where further substitutions should be avoided either alone or in combination with other mutations.

[0191] A number of scientific publications have been devoted to the prediction of secondary structure. See Moult J., Curr. Op. in Biotech., 7(4): 422-427 (1996), Chou et al., Biochemistry, 13(2): 222-245 (1974); Chou et al., Biochemistry, 113(2): 211-222 (1974); Chou et al., Adv. Enzymol. Relat. Areas Mol. Biol., 47: 45-148 (1978); Chou et al., Ann. Rev. Biochem., 47: 251-276 and Chou et al., Biophys. J., 26: 367-384 (1979). Moreover, computer programs are currently available to assist with predicting secondary structure. One method of predicting secondary structure is based upon homology modeling. For example, two polypeptides or proteins which have a sequence identity of greater than 30%, or similarity greater than 40% often have similar structural topologies. The recent growth of the protein structural data base (PDB) has provided enhanced predictability of secondary structure, including the potential number of folds within a polypeptide's or protein's structure. See Holm et al., Nucl. Acid. Res., 27(1): 244-247 (1999). It has been suggested (Brenner et al. Curr. Op. Struct. Biol., 7(3): 369-376 (1997)) that there are a limited number of folds in a given polypeptide or protein and that once a critical number of structures have been resolved, structural prediction will gain dramatically in accuracy.

[0192] Additional methods of predicting secondary structure include “threading” (Jones, D., Curr. Opin. Struct. Biol., 7(3): 377-87 (1997); Sippl et al., Structure, 4(1): 15-9 (1996)), “profile analysis” (Bowie et al., Science, 253: 164-170 (1991); Gribskov et al., Meth. Enzym., 183: 146-159 (1990); Gribskov et al., Proc. Nat. Acad. Sci., 84(13): 4355-8 (1987)), and “evolutionary linkage” (See Home, supra, and Brenner, supra).

[0193] Vehicles. This invention requires the presence of at least one vehicle (V¹) attached to a peptide through the N-terminus, C-terminus or a sidechain of one of the amino acid residues. Multiple vehicles may also be used; e.g., Fc's at each terminus or an Fc at a terminus and a PEG group at the other terminus or a sidechain. Exemplary vehicles include:

[0194] an Fc domain;

[0195] other proteins, polypeptides, or peptides capable of binding to a salvage receptor;

[0196] human serum albumin (HSA);

[0197] a leucine zipper (LZ) domain;

[0198] polyethylene glycol (PEG), including 5 kD, 20 kD, and 30 kD PEG, as well as other polymers;

[0199] dextran;

[0200] and other molecules known in the art to provide extended half-life and/or protection from proteolytic degradation or clearance.

[0201] An Fc domain is the preferred vehicle. The Fc domain may be fused to the N or C termini of the peptides or at both the N and C termini. Fusion to the N terminus is preferred.

[0202] As noted above, Fc variants are suitable vehicles within the scope of this invention. A native Fc may be extensively modified to form an Fc variant in accordance with this invention, provided binding to the salvage receptor is maintained; see, for example WO 97/34631 and WO 96/32478. In such Fc variants, one may remove one or more sites of a native Fc that provide structural features or functional activity not required by the fusion molecules of this invention. One may remove these sites by, for example, substituting or deleting residues, inserting residues into the site, or truncating portions containing the site. The inserted or substituted residues may also be altered amino acids, such as peptidomimetics or D-amino acids. Fc variants may be desirable for a number of reasons, several of which are described below. Exemplary Fc variants include molecules and sequences in which:

[0203] 1. Sites involved in disulfide bond formation are removed. Such removal may avoid reaction with other cysteine-containing proteins present in the host cell used to produce the molecules of the invention. For this purpose, the cysteine-containing segment at the N-terminus may be truncated or cysteine residues may be deleted or substituted with other amino acids (e.g., alanyl, seryl). In particular, one may truncate the N-terminal 20-amino acid segment of SEQ ID NO: 2 or delete or substitute the cysteine residues at positions 7 and 10 of SEQ ID NO: 2. Even when cysteine residues are removed, the single chain Fc domains can still form a dimeric Fc domain that is held together non-covalently.

[0204] 2. A native Fc is modified to make it more compatible with a selected host cell. For example, one may remove the PA sequence near the N-terminus of a typical native Fc, which may be recognized by a digestive enzyme in E. coli such as proline iminopeptidase. One may also add an N-terminal methionine residue, especially when the molecule is expressed recombinantly in a bacterial cell such as E. coli. The Fc domain of SEQ ID NO: 2 is one such Fc variant.

[0205] 3. A portion of the N-terminus of a native Fc is removed to prevent N-terminal heterogeneity when expressed in a selected host cell. For this purpose, one may delete any of the first 20 amino acid residues at the N-terminus, particularly those at positions 1, 2, 3, 4 and 5.

[0206]4. One or more glycosylation sites are removed. Residues that are typically glycosylated (e.g., asparagine) may confer cytolytic response. Such residues may be deleted or substituted with unglycosylated residues (e.g., alanine).

[0207] 5. Sites involved in interaction with complement, such as the C1q binding site, are removed. For example, one may delete or substitute the EKK sequence of human IgG1. Complement recruitment may not be advantageous for the molecules of this invention and so may be avoided with such an Fc variant.

[0208] 6. Sites are removed that affect binding to Fc receptors other than a salvage receptor. A native Fc may have sites for interaction with certain white blood cells that are not required for the fusion molecules of the present invention and so may be removed.

[0209] 7. The ADCC site is removed. ADCC sites are known in the art; see, for example, Molec. Immunol. 29 (5): 633-9 (1992) with regard to ADCC sites in IgG1. These sites, as well, are not required for the fusion molecules of the present invention and so may be removed.

[0210] 8. When the native Fc is derived from a non-human antibody, the native Fc may be humanized. Typically, to humanize a native Fc, one will substitute selected residues in the non-human native Fc with residues that are normally found in human native Fc. Techniques for antibody humanization are well known in the art.

[0211] Preferred Fc variants include the following. In SEQ ID NO: 2 (FIG. 3), the leucine at position 15 may be substituted with glutamate; the glutamate at position 99, with alanine; and the lysines at positions 101 and 103, with alanines. In addition, one or more tyrosine residues can be replaced by phenyalanine residues.

[0212] An alternative vehicle would be a protein, polypeptide, peptide, antibody, antibody fragment, or small molecule (e.g., a peptidomimetic compound) capable of binding to a salvage receptor. For example, one could use as a vehicle a polypeptide as described in U.S. Pat. No. 5,739,277, issued Apr. 14, 1998 to Presta et al. Peptides could also be selected by phage display or RNA-peptide screening for binding to the FcRn salvage receptor. Such salvage receptor-binding compounds are also included within the meaning of “vehicle” and are within the scope of this invention. Such vehicles should be selected for increased half-life (e.g., by avoiding sequences recognized by proteases) and decreased immunogenicity (e.g., by favoring non-immunogenic sequences, as discovered in antibody humanization).

[0213] As noted above, polymer vehicles may also be used for V¹. Various means for attaching chemical moieties useful as vehicles are currently available, see, e.g., Patent Cooperation Treaty (“PCT”) International Publication No. WO 96/11953, entitled “N-Terminally Chemically Modified Protein Compositions and Methods,” herein incorporated by reference in its entirety. This PCT publication discloses, among other things, the selective attachment of water soluble polymers to the N-terminus of proteins.

[0214] A preferred polymer vehicle is polyethylene glycol (PEG). The PEG group may be of any convenient molecular weight and may be linear or branched. The average molecular weight of the PEG will preferably range from about 2 kiloDalton (“kD”) to about 100 kD, more preferably from about 5 kD to about 50 kD, most preferably from about 5 kD to about 10 kD. The PEG groups will generally be attached to the compounds of the invention via acylation or reductive alkylation through a reactive group on the PEG moiety (e.g., an aldehyde, amino, thiol, or ester group) to a reactive group on the inventive compound (e.g., an aldehyde, amino, or ester group).

[0215] A useful strategy for the PEGylation of synthetic peptides consists of combining, through forming a conjugate linkage in solution, a peptide and a PEG moiety, each bearing a special functionality that is mutually reactive toward the other. The peptides can be easily prepared with conventional solid phase synthesis. The peptides are “preactivated” with an appropriate functional group at a specific site. The precursors are purified and fully characterized prior to reacting with the PEG moiety. Ligation of the peptide with PEG usually takes place in aqueous phase and can be easily monitored by reverse phase analytical HPLC. The PEGylated peptides can be easily purified by preparative HPLC and characterized by analytical HPLC, amino acid analysis and laser desorption mass spectrometry.

[0216] Polysaccharide polymers are another type of water soluble polymer which may be used for protein modification. Dextrans are polysaccharide polymers comprised of individual subunits of glucose predominantly linked by α1-6 linkages. The dextran itself is available in many molecular weight ranges, and is readily available in molecular weights from about 1 kD to about 70 kD. Dextran is a suitable water soluble polymer for use in the present invention as a vehicle by itself or in combination with another vehicle (e.g., Fc). See, for example, WO 96/11953 and WO 96/05309. The use of dextran conjugated to therapeutic or diagnostic immunoglobulins has been reported; see, for example, European Patent Publication No. 0 315 456, which is hereby incorporated by reference in its entirety. Dextran of about 1 kD to about 20 kD is preferred when dextran is used as a vehicle in accordance with the present invention.

[0217] Linkers. Any “linker” group is optional. When present, its chemical structure is not critical, since it serves primarily as a spacer. The linker is preferably made up of amino acids linked together by peptide bonds. Thus, in preferred embodiments, the linker is made up of from 1 to 30 amino acids linked by peptide bonds, wherein the amino acids are selected from the 20 naturally occurring amino acids. Some of these amino acids may be glycosylated, as is well understood by those in the art. In a more preferred embodiment, the 1 to 20 amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. Even more preferably, a linker is made up of a majority of amino acids that are sterically unhindered, such as glycine and alanine. Thus, preferred linkers are polyglycines (particularly (Gly)₄, (Gly)₅), poly(Gly-Ala), and polyalanines. Other specific examples of linkers are: (Gly)₃Lys(Gly)₄; (SEQ ID NO: 40) (Gly)₃AsnGlySer(Gly)₂; (SEQ ID NO: 41) (Gly)₃Cys(Gly)₄; and (SEQ ID NO: 42) GlyProAsnGlyGly. (SEQ ID NO: 43)

[0218] To explain the above nomenclature, for example, (Gly)₃Lys(Gly)₄ means Gly-Gly-Gly-Lys-Gly-Gly-Gly-Gly (SEQ ID NO: 40). Combinations of Gly and Ala are also preferred. The linkers shown here are exemplary; linkers within the scope of this invention may be much longer and may include other residues.

[0219] Preferred linkers are amino acid linkers comprising greater than 5 amino acids, with suitable linkers having up to about 500 amino acids selected from glycine, alanine, proline, asparagine, glutamine, lysine, threonine, serine or aspartate. Linkers of about 20 to 50 amino acids are most preferred. One group of preferred linkers are those of the formulae (SEQ ID NO: 193) GSGSATGGSGSTASSGSGSATx¹x² and (SEQ ID NO: 194) GSGSATGGSGSTASSGSGSATx¹x²GSGSATGGSGSTASSGSGSATx³x⁴

[0220] wherein x¹ and x³ are each independently basic or hydrophobic residues and x² and X⁴ are each independently hydrophobic residues. Specific preferred linkers are: (SEQ ID NO: 59) GSGSATGGSGSTASSGSGSATHM (SEQ ID NO: 190) GSGSATGGSGSTASSGSGSATGM (SEQ ID NO: 191) GSGSATGGSGSTASSGSGSATGS, and (SEQ ID NO: 192) GSGSATGGSGSTASSGSGSATHMGSGSATGGSGSTASSGSGSATHM.

[0221] Non-peptide linkers are also possible. For example, alkyl linkers such as —NH—(CH₂)_(s)—C(O)—, wherein s=2-20 could be used. These alkyl linkers may further be substituted by any non-sterically hindering group such as lower alkyl (e.g., C₁—C₆) lower acyl, halogen (e.g., Cl, Br), CN, NH₂, phenyl, etc. An exemplary non-peptide linker is a PEG linker,

[0222] wherein n is such that the linker has a molecular weight of 100 to 5000 kD, preferably 100 to 500 kD. The peptide linkers may be altered to form derivatives in the same manner as described above.

[0223] Derivatives. The inventors also contemplate derivatizing the peptide and/or vehicle portion of the compounds. Such derivatives may improve the solubility, absorption, biological half life, and the like of the compounds. The moieties may alternatively eliminate or attenuate any undesirable side-effect of the compounds and the like. Exemplary derivatives include compounds in which:

[0224] 1. The compound or some portion thereof is cyclic. For example, the peptide portion may be modified to contain two or more Cys residues (e.g., in the linker), which could cyclize by disulfide bond formation.

[0225] 2. The compound is cross-linked or is rendered capable of cross-linking between molecules. For example, the peptide portion may be modified to contain one Cys residue and thereby be able to form an intermolecular disulfide bond with a like molecule. The compound may also be cross-linked through its C-terminus, as in the molecule shown below.

[0226] In Formula VIII, each “V¹” may represent typically one strand of the Fc domain.

[0227] 3. One or more peptidyl [—C(O)NR—] linkages (bonds) is replaced by a non-peptidyl linkage. Exemplary non-peptidyl linkages are —CH₂— carbamate [—CH₂—OC(O)NR—], phosphonate, —CH₂-sulfonamide [—CH₂—S(O)₂NR—], urea [—NHC(O)NH—], —CH₂-secondary amine, and alkylated peptide [—C(O)NR⁶— wherein R⁶ is lower alkyl].

[0228] 4. The N-terminus is derivatized. Typically, the N-terminus may be acylated or modified to a substituted amine. Exemplary N-terminal derivative groups include —NRR¹ (other than —NH₂), —NRC(O)R¹, —NRC(O)OR¹, —NRS(O)₂R¹, —NHC(O)NHR¹, succinimide, or benzyloxycarbonyl-NH—(CBZ—NH—), wherein R and R¹ are each independently hydrogen or lower alkyl and wherein the phenyl ring may be substituted with 1 to 3 substituents selected from the group consisting of C₁-C₄ alkyl, C₁-C₄ alkoxy, chloro, and bromo.

[0229] 5. The free C-terminus is derivatized. Typically, the C-terminus is esterified or amidated. Exemplary C-terminal derivative groups include, for example, —C(O)R² wherein R² is lower alkoxy or —NR³R⁴ wherein R³ and R⁴ are independently hydrogen or C₁-C₈ alkyl (preferably C₁-C₄ alkyl).

[0230] 6. A disulfide bond is replaced with another, preferably more stable, cross-linking moiety (e.g., an alkylene). See, e.g., Bhatnagar et al. (1996), J. Med. Chem. 39: 3814-9; Alberts et al. (1993) Thirteenth Am. Pep. Symp., 357-9.

[0231] 7. One or more individual amino acid residues is modified. Various derivatizing agents are known to react specifically with selected sidechains or terminal residues, as described in detail below.

[0232] Lysinyl residues and amino terminal residues may be reacted with succinic or other carboxylic acid anhydrides, which reverse the charge of the lysinyl residues. Other suitable reagents for derivatizing alpha-amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate.

[0233] Arginyl residues may be modified by reaction with any one or combination of several conventional reagents, including phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginyl residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.

[0234] Specific modification of tyrosyl residues has been studied extensively, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.

[0235] Carboxyl sidechain groups (aspartyl or glutamyl) may be selectively modified by reaction with carbodiimides (R′—N═C═N—R′) such as 1-cyclohexyl-3-(2-morpholinyl-(4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore, aspartyl and glutamyl residues may be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

[0236] Glutaminyl and asparaginyl residues may be deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention.

[0237] Cysteinyl residues can be replaced by amino acid residues or other moieties either to eliminate disulfide bonding or, conversely, to stabilize cross-linking. See, e.g., Bhatnagar et al. (1996), J. Med. Chem. 39: 3814-9.

[0238] Derivatization with bifunctional agents is useful for cross-linking the peptides or their functional derivatives to a water-insoluble support matrix or to other macromolecular vehicles. Commonly used cross-linking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane. Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates that are capable of forming cross-links in the presence of light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are employed for protein immobilization.

[0239] Carbohydrate (oligosaccharide) groups may conveniently be attached to sites that are known to be glycosylation sites in proteins. Generally, O-linked oligosaccharides are attached to serine (Ser) or threonine (Thr) residues while N-linked oligosaccharides are attached to asparagine (Asn) residues when they are part of the sequence Asn-X-Ser/Thr, where X can be any amino acid except proline. X is preferably one of the 19 naturally occurring amino acids other than proline. The structures of N-linked and O-linked oligosaccharides and the sugar residues found in each type are different. One type of sugar that is commonly found on both is N-acetylneuraminic acid (referred to as sialic acid). Sialic acid is usually the terminal residue of both N-linked and O-linked oligosaccharides and, by virtue of its negative charge, may confer acidic properties to the glycosylated compound. Such site(s) may be incorporated in the linker of the compounds of this invention and are preferably glycosylated by a cell during recombinant production of the polypeptide compounds (e.g., in mammalian cells such as CHO, BHK, COS). However, such sites may further be glycosylated by synthetic or semi-synthetic procedures known in the art.

[0240] Other possible modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, oxidation of the sulfur atom in Cys, methylation of the alpha-amino groups of lysine, arginine, and histidine side chains. Creighton, Proteins: Structure and Molecule Properties (W. H. Freeman & Co., San Francisco), pp. 79-86 (1983).

[0241] Compounds of the present invention may be changed at the DNA level, as well. The DNA sequence of any portion of the compound may be changed to codons more compatible with the chosen host cell. For E. coli, which is the preferred host cell, optimized codons are known in the art. Codons may be substituted to eliminate restriction sites or to include silent restriction sites, which may aid in processing of the DNA in the selected host cell. The vehicle, linker and peptide DNA sequences may be modified to include any of the foregoing sequence changes.

[0242] Methods of Making

[0243] The compounds of this invention largely may be made in transformed host cells using recombinant DNA techniques. To do so, a recombinant DNA molecule coding for the peptide is prepared. Methods of preparing such DNA molecules are well known in the art. For instance, sequences coding for the peptides could be excised from DNA using suitable restriction enzymes. Alternatively, the DNA molecule could be synthesized using chemical synthesis techniques, such as the phosphoramidate method. Also, a combination of these techniques could be used.

[0244] The invention also includes a vector capable of expressing the peptides in an appropriate host. The vector comprises the DNA molecule that codes for the peptides operatively linked to appropriate expression control sequences. Methods of effecting this operative linking, either before or after the DNA molecule is inserted into the vector, are well known. Expression control sequences include promoters, activators, enhancers, operators, ribosomal binding sites, start signals, stop signals, cap signals, polyadenylation signals, and other signals involved with the control of transcription or translation.

[0245] The resulting vector having the DNA molecule thereon is used to transform an appropriate host. This transformation may be performed using methods well known in the art.

[0246] Any of a large number of available and well-known host cells may be used in the practice of this invention. The selection of a particular host is dependent upon a number of factors recognized by the art. These include, for example, compatibility with the chosen expression vector, toxicity of the peptides encoded by the DNA molecule, rate of transformation, ease of recovery of the peptides, expression characteristics, bio-safety and costs. A balance of these factors must be struck with the understanding that not all hosts may be equally effective for the expression of a particular DNA sequence. Within these general guidelines, useful microbial hosts include bacteria (such as E. coli sp.), yeast (such as Saccharomyces sp.) and other fungi, insects, plants, mammalian (including human) cells in culture, or other hosts known in the art.

[0247] Next, the transformed host is cultured and purified. Host cells may be cultured under conventional fermentation conditions so that the desired compounds are expressed. Such fermentation conditions are well known in the art. Finally, the peptides are purified from culture by methods well known in the art.

[0248] The compounds may also be made by synthetic methods. For example, solid phase synthesis techniques may be used. Suitable techniques are well known in the art, and include those described in Merrifield (1973), Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotis eds.); Merrifield (1963), J. Am. Chem. Soc. 85: 2149; Davis et al. (1985), Biochem. Intl. 10: 394-414; Stewart and Young (1969), Solid Phase Peptide Synthesis; U.S. Pat. No. 3,941,763; Finn et al. (1976), The Proteins (3rd ed.) 2: 105-253; and Erickson et al. (1976), The Proteins (3rd ed.) 2: 257-527. Solid phase synthesis is the preferred technique of making individual peptides since it is the most cost-effective method of making small peptides.

[0249] Compounds that contain derivatized peptides or which contain non-peptide groups may be synthesized by well-known organic chemistry techniques.

[0250] Uses of the Compounds

[0251] Compounds of this invention may be particularly useful in treatment of B-cell mediated autoimmune diseases. In particular, the compounds of this invention may be useful in treating, preventing, ameliorating, diagnosing or prognosing lupus, including systemic lupus erythematosus (SLE), and lupus-associated diseases and conditions. Other preferred indications include B-cell mediated cancers, including B-cell lymphoma.

[0252] The compounds of this invention can also be used to treat inflammatory conditions of the joints. Inflammatory conditions of a joint are chronic joint diseases that afflict and disable, to varying degrees, millions of people worldwide. Rheumatoid arthritis is a disease of articular joints in which the cartilage and bone are slowly eroded away by a proliferative, invasive connective tissue called pannus, which is derived from the synovial membrane. The disease may involve peri-articular structures such as bursae, tendon sheaths and tendons as well as extra-articular tissues such as the subcutis, cardiovascular system, lungs, spleen, lymph nodes, skeletal muscles, nervous system (central and peripheral) and eyes (Silberberg (1985), Anderson's Pathology, Kissane (ed.), II:1828). Osteoarthritis is a common joint disease characterized by degenerative changes in articular cartilage and reactive proliferation of bone and cartilage around the joint. Osteoarthritis is a cell-mediated active process that may result from the inappropriate response of chondrocytes to catabolic and anabolic stimuli. Changes in some matrix molecules of articular cartilage reportedly occur in early osteoarthritis (Thonar et al. (1993), Rheumatic disease clinics of North America, Moskowitz (ed.), 19:635-657 and Shinmei et al. (1992), Arthritis Rheum., 35:1304-1308). TALL-1, TALL-1R and modulators thereof are believed to be useful in the treatment of these and related conditions.

[0253] Compounds of this invention may also be useful in treatment of a number of additional diseases and disorders, including:

[0254] acute pancreatitis;

[0255] ALS;

[0256] Alzheimer's disease;

[0257] asthma;

[0258] atherosclerosis;

[0259] autoimmune hemolytic anemia;

[0260] cancer, particularly cancers related to B cells;

[0261] cachexia/anorexia;

[0262] chronic fatigue syndrome;

[0263] cirrhosis (e.g., primary biliary cirrhosis);

[0264] diabetes (e.g., insulin diabetes);

[0265] fever;

[0266] glomerulonephritis, including IgA glomerulonephritis and primary glomerulonephritis;

[0267] Goodpasture's syndrome;

[0268] Guillain-Barre syndrome;

[0269] graft versus host disease;

[0270] Hashimoto's thyroiditis;

[0271] hemorrhagic shock;

[0272] hyperalgesia;

[0273] inflammatory bowel disease;

[0274] inflammatory conditions of a joint, including osteoarthritis, psoriatic arthritis and rheumatoid arthritis;

[0275] inflammatory conditions resulting from strain, sprain, cartilage damage, trauma, orthopedic surgery, infection or other disease processes;

[0276] insulin-dependent diabetes mellitus;

[0277] ischemic injury, including cerebral ischemia (e.g., brain injury as a result of trauma, epilepsy, hemorrhage or stroke, each of which may lead to neurodegeneration);

[0278] learning impairment;

[0279] lung diseases (e.g., ARDS);

[0280] multiple myeloma;

[0281] multiple sclerosis;

[0282] Myasthenia gravis;

[0283] myelogenous (e.g., AML and CML) and other leukemias;

[0284] myopathies (e.g., muscle protein metabolism, esp. in sepsis);

[0285] neurotoxicity (e.g., as induced by HIV);

[0286] osteoporosis;

[0287] pain;

[0288] Parkinson's disease;

[0289] Pemphigus;

[0290] polymyositis/dermatomyositis;

[0291] pulmonary inflammation, including autoimmune pulmonary inflammation;

[0292] pre-term labor;

[0293] psoriasis;

[0294] Reiter's disease;

[0295] reperfusion injury;

[0296] septic shock;

[0297] side effects from radiation therapy;

[0298] Sjogren's syndrome;

[0299] sleep disturbance;

[0300] temporal mandibular joint disease;

[0301] thrombocytopenia, including idiopathic thrombocytopenia and autoimmune neonatal thrombocytopenia;

[0302] tumor metastasis;

[0303] uveitis; and

[0304] vasculitis.

[0305] Compounds of this invention may be administered alone or in combination with a therapeutically effective amount of other drugs, including analgesic agents, disease-modifying anti-rheumatic drugs (DMARDs), non-steroidal anti-inflammatory drugs (NSAIDs), and any immune and/or inflammatory modulators. Thus, compounds of this invention may be administered with:

[0306] Modulators of other members of the TNF/TNF receptor family, including TNF antagonists, such as etanercept (Enbrel™), sTNF-RI, onercept, D2E7, and Remicade™.

[0307] Nerve growth factor (NGF) modulators.

[0308] IL-1 inhibitors, including IL-1ra molecules such as anakinra and more recently discovered IL-1ra-like molecules such as IL-1Hy1 and IL-1Hy2; IL-1 “trap” molecules as described in U.S. Pat. No. 5,844,099, issued Dec. 1, 1998; IL-1 antibodies; solubilized IL-1 receptor, and the like.

[0309] IL-6 inhibitors (e.g., antibodies to IL-6).

[0310] IL-8 inhibitors (e.g., antibodies to IL-8).

[0311] IL-18 inhibitors (e.g., IL-18 binding protein, solubilized IL-18 receptor, or IL-18 antibodies).

[0312] Interleukin-1 converting enzyme (ICE) modulators.

[0313] insulin-like growth factors (IGF-1, IGF-2) and modulators thereof.

[0314] Transforming growth factor-β (TGF-β), TGF-β family members, and TGF-β modulators.

[0315] Fibroblast growth factors FGF-1 to FGF-10, and FGF modulators.

[0316] Osteoprotegerin (OPG), OPG analogues, osteoprotective agents, and antibodies to OPG-ligand (OPG-L).

[0317] bone anabolic agents, such as parathyroid hormone (PTH), PTH fragments, and molecules incorporating PTH fragments (e.g., PTH (1-34)-Fc).

[0318] PAF antagonists.

[0319] Keratinocyte growth factor (KGF), KGF-related molecules (e.g., KGF-2), and KGF modulators.

[0320] COX-2 inhibitors, such as Celebrex™ and Vioxx™.

[0321] Prostaglandin analogs (e.g., E series prostaglandins).

[0322] Matrix metalloproteinase (MMP) modulators.

[0323] Nitric oxide synthase (NOS) modulators, including modulators of inducible NOS.

[0324] Modulators of glucocorticoid receptor.

[0325] Modulators of glutamate receptor.

[0326] Modulators of lipopolysaccharide (LPS) levels.

[0327] Anti-cancer agents, including inhibitors of oncogenes (e.g., fos, jun) and interferons.

[0328] Noradrenaline and modulators and mimetics thereof.

[0329] Pharmaceutical Compositions

[0330] In General. The present invention also provides methods of using pharmaceutical compositions of the inventive compounds. Such pharmaceutical compositions may be for administration for injection, or for oral, pulmonary, nasal, transdermal or other forms of administration. In general, the invention encompasses pharmaceutical compositions comprising effective amounts of a compound of the invention together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. Such compositions include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol); incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes. Hyaluronic acid may also be used, and this may have the effect of promoting sustained duration in the circulation. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present proteins and derivatives. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712 which are herein incorporated by reference in their entirety. The compositions may be prepared in liquid form, or may be in dried powder, such as lyophilized form. Implantable sustained release formulations are also contemplated, as are transdermal formulations.

[0331] Oral dosage forms. Contemplated for use herein are oral solid dosage forms, which are described generally in Chapter 89 of Remington's Pharmaceutical Sciences (1990), 18th Ed., Mack Publishing Co. Easton Pa. 18042, which is herein incorporated by reference in its entirety. Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets or pellets. Also, liposomal or proteinoid encapsulation may be used to formulate the present compositions (as, for example, proteinoid microspheres reported in U.S. Pat. No. 4,925,673). Liposomal encapsulation may be used and the liposomes may be derivatized with various polymers (e.g., U.S. Pat. No. 5,013,556). A description of possible solid dosage forms for the therapeutic is given in Chapter 10 of Marshall, K., Modern Pharmaceutics (1979), edited by G. S. Banker and C. T. Rhodes, herein incorporated by reference in its entirety. In general, the formulation will include the inventive compound, and inert ingredients which allow for protection against the stomach environment, and release of the biologically active material in the intestine.

[0332] Also specifically contemplated are oral dosage forms of the above inventive compounds. If necessary, the compounds may be chemically modified so that oral delivery is efficacious. Generally, the chemical modification contemplated is the attachment of at least one moiety to the compound molecule itself, where said moiety permits (a) inhibition of proteolysis; and (b) uptake into the blood stream from the stomach or intestine. Also desired is the increase in overall stability of the compound and increase in circulation time in the body. Moieties useful as covalently attached vehicles in this invention may also be used for this purpose. Examples of such moieties include: PEG, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline. See, for example, Abuchowski and Davis, Soluble Polymer-Enzyme Adducts, Enzymes as Drugs (1981), Hocenberg and Roberts, eds., Wiley-Interscience, New York, N.Y., pp. 367-83; Newmark, et al. (1982), J. Appl. Biochem. 4:185-9. Other polymers that could be used are poly-1,3-dioxolane and poly-1,3,6-tioxocane. Preferred for pharmaceutical usage, as indicated above, are PEG moieties.

[0333] For oral delivery dosage forms, it is also possible to use a salt of a modified aliphatic amino acid, such as sodium N-(8-[2-hydroxybenzoyl]amino) caprylate (SNAC), as a carrier to enhance absorption of the therapeutic compounds of this invention. The clinical efficacy of a heparin formulation using SNAC has been demonstrated in a Phase II trial conducted by Emisphere Technologies. See U.S. Pat. No. 5,792,451, “Oral drug delivery composition and methods”.

[0334] The compounds of this invention can be included in the formulation as fine multiparticulates in the form of granules or pellets of particle size about 1 mm. The formulation of the material for capsule administration could also be as a powder, lightly compressed plugs or even as tablets. The therapeutic could be prepared by compression.

[0335] Colorants and flavoring agents may all be included. For example, the protein (or derivative) may be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage containing colorants and flavoring agents.

[0336] One may dilute or increase the volume of the compound of the invention with an inert material. These diluents could include carbohydrates, especially mannitol, α-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch. Certain inorganic salts may also be used as fillers including calcium triphosphate, magnesium carbonate and sodium chloride. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.

[0337] Disintegrants may be included in the formulation of the therapeutic into a solid dosage form. Materials used as disintegrants include but are not limited to starch including the commercial disintegrant based on starch, Explotab. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite may all be used. Another form of the disintegrants are the insoluble cationic exchange resins. Powdered gums may be used as disintegrants and as binders and these can include powdered gums such as agar, Karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants.

[0338] Binders may be used to hold the therapeutic agent together to form a hard tablet and include materials from natural products such as acacia, tragacanth, starch and gelatin. Others include methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both be used in alcoholic solutions to granulate the therapeutic.

[0339] An antifrictional agent may be included in the formulation of the therapeutic to prevent sticking during the formulation process. Lubricants may be used as a layer between the therapeutic and the die wall, and these can include but are not limited to; stearic acid including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants may also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000.

[0340] Glidants that might improve the flow properties of the drug during formulation and to aid rearrangement during compression might be added. The glidants may include starch, talc, pyrogenic silica and hydrated silicoaluminate.

[0341] To aid dissolution of the compound of this invention into the aqueous environment a surfactant might be added as a wetting agent. Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents might be used and could include benzalkonium chloride or benzethonium chloride. The list of potential nonionic detergents that could be included in the formulation as surfactants are lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants could be present in the formulation of the protein or derivative either alone or as a mixture in different ratios.

[0342] Additives may also be included in the formulation to enhance uptake of the compound. Additives potentially having this property are for instance the fatty acids oleic acid, linoleic acid and linolenic acid.

[0343] Controlled release formulation may be desirable. The compound of this invention could be incorporated into an inert matrix which permits release by either diffusion or leaching mechanisms; e.g., gums. Slowly degenerating matrices may also be incorporated into the formulation, e.g., alginates, polysaccharides. Another form of a controlled release of the compounds of this invention is by a method based on the Oros therapeutic system (Alza Corp.), i.e., the drug is enclosed in a semipermeable membrane which allows water to enter and push drug out through a single small opening due to osmotic effects. Some enteric coatings also have a delayed release effect.

[0344] Other coatings may be used for the formulation. These include a variety of sugars which could be applied in a coating pan. The therapeutic agent could also be given in a film coated tablet and the materials used in this instance are divided into 2 groups. The first are the nonenteric materials and include methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose, providone and the polyethylene glycols. The second group consists of the enteric materials that are commonly esters of phthalic acid.

[0345] A mix of materials might be used to provide the optimum film coating. Film coating may be carried out in a pan coater or in a fluidized bed or by compression coating.

[0346] Pulmonary delivery forms. Also contemplated herein is pulmonary delivery of the present protein (or derivatives thereof). The protein (or derivative) is delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining to the blood stream. (Other reports of this include Adjei et al., Pharma. Res. (1990) 7: 565-9; Adjei et al. (1990), Internatl. J. Pharmaceutics 63: 135-44 (leuprolide acetate); Braquet et al. (1989), J. Cardiovasc. Pharmacol. 13 (suppl.5): s.143-146 (endothelin-1); Hubbard et al. (1989), Annals Int. Med. 3: 206-12 (α1-antitrypsin); Smith et al. (1989), J. Clin. Invest. 84: 1145-6 (α1-proteinase); Oswein et al. (March 1990), “Aerosolization of Proteins”, Proc. Symp. Resp. Drug Delivery II, Keystone, Colo. (recombinant human growth hormone); Debs et al. (1988), J. Immunol. 140: 3482-8 (interferon-γ and tumor necrosis factor α) and Platz et al, U.S. Pat. No. 5,284,656 (granulocyte colony stimulating factor).

[0347] Contemplated for use in the practice of this invention are a wide range of mechanical devices designed for pulmonary delivery of therapeutic products, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. Some specific examples of commercially available devices suitable for the practice of this invention are the Ultravent nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the Acorn II nebulizer, manufactured by Marquest Medical Products, Englewood, Colo.; the Ventolin metered dose inhaler, manufactured by Glaxo Inc., Research Triangle Park, N.C.; and the Spinhaler powder inhaler, manufactured by Fisons Corp., Bedford, Mass.

[0348] All such devices require the use of formulations suitable for the dispensing of the inventive compound. Typically, each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in addition to diluents, adjuvants and/or carriers useful in therapy.

[0349] The inventive compound should most advantageously be prepared in particulate form with an average particle size of less than 10 μm (or microns), most preferably 0.5 to 5 μm, for most effective delivery to the distal lung.

[0350] Pharmaceutically acceptable carriers include carbohydrates such as trehalose, mannitol, xylitol, sucrose, lactose, and sorbitol. Other ingredients for use in formulations may include DPPC, DOPE, DSPC and DOPC. Natural or synthetic surfactants may be used. PEG may be used (even apart from its use in derivatizing the protein or analog). Dextrans, such as cyclodextran, may be used. Bile salts and other related enhancers may be used. Cellulose and cellulose derivatives may be used. Amino acids may be used, such as use in a buffer formulation.

[0351] Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated.

[0352] Formulations suitable for use with a nebulizer, either jet or ultrasonic, will typically comprise the inventive compound dissolved in water at a concentration of about 0.1 to 25 mg of biologically active protein per mL of solution. The formulation may also include a buffer and a simple sugar (e.g., for protein stabilization and regulation of osmotic pressure). The nebulizer formulation may also contain a surfactant, to reduce or prevent surface induced aggregation of the protein caused by atomization of the solution in forming the aerosol.

[0353] Formulations for use with a metered-dose inhaler device will generally comprise a finely divided powder containing the inventive compound suspended in a propellant with the aid of a surfactant. The propellant may be any conventional material employed for this purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactants include sorbitan trioleate and soya lecithin. Oleic acid may also be useful as a surfactant.

[0354] Formulations for dispensing from a powder inhaler device will comprise a finely divided dry powder containing the inventive compound and may also include a bulking agent, such as lactose, sorbitol, sucrose, mannitol, trehalose, or xylitol in amounts which facilitate dispersal of the powder from the device, e.g., 50 to 90% by weight of the formulation.

[0355] Nasal delivery forms. Nasal delivery of the inventive compound is also contemplated. Nasal delivery allows the passage of the protein to the blood stream directly after administering the therapeutic product to the nose, without the necessity for deposition of the product in the lung. Formulations for nasal delivery include those with dextran or cyclodextran. Delivery via transport across other mucous membranes is also contemplated.

[0356] Dosages. The dosage regimen involved in a method for treating the above-described conditions will be determined by the attending physician, considering various factors which modify the action of drugs, e.g. the age, condition, body weight, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors. Generally, the daily regimen should be in the range of 0.1-1000 micrograms of the inventive compound per kilogram of body weight, preferably 0.1-150 micrograms per kilogram.

[0357] Specific Preferred Embodiments

[0358] The inventors have determined preferred structures for the preferred peptides listed in Table 4 below. The symbol “Λ” may be any of the linkers described herein or may simply represent a normal peptide bond (i.e., so that no linker is present). Tandem repeats and linkers are shown separated by dashes for clarity. TABLE 4 Preferred embodiments SEQ ID Sequence/structure NO: LPGCKWDLLIKQWVCDPL-Λ-V¹ 44 V¹-Λ- LPGCKWDLLIKQWVCDPL 45 LPGCKWDLLIKQWVCDPL -Λ- 46 LPGCKWDLLIKQWVCDPL -Λ-V¹ V¹-Λ- LPGCKWDLLIKQWVCDPL -Λ- 47 LPGCKWDLLIKQWVCDPL SADCYFDILTKSDVCTSS-Λ-V¹ 48 V¹-Λ- SADCYFDILTKSDVCTSS 49 SADCYFDILTKSDVTSS-Λ- SADCYFDILTKSDVTSS 50 -Λ-V¹ V¹-Λ- SADCYFDILTKSDVTSS -Λ- 51 SADCYFDILTKSDVTSS FHDCKWDLLTKQWVCHGL-Λ-V¹ 52 V¹-Λ- FHDCKWDLLTKQWVCHGL 53 FHDCKWDLLTKQWVCHGL -Λ- 54 FHDCKWDLLTKQWVCHGL -Λ-V¹ V¹-Λ- FHDCKWDLLTKQWVCHGL -Λ- 55 FHDCKWDLLTKQWVCHGL

[0359] “V¹” is an Fc domain as defined previously herein. In addition to those listed in Table 4, the inventors further contemplate heterodimers in which each strand of an Fc dimer is linked to a different peptide sequence; for example, wherein each Fc is linked to a different sequence selected from Table 2.

[0360] All of the compounds of this invention can be prepared by methods described in PCT appl. no. WO 99/25044.

[0361] The invention will now be further described by the following working examples, which are illustrative rather than limiting.

EXAMPLE 1 Peptides

[0362] Peptide Phage Display

[0363] 1. Magnetic Bead Preparation

[0364] A. Fc-TALL-1 Immobilization on Magnetic Beads

[0365] The recombinant Fc-TALL-1 protein was immobilized on the Protein A Dynabeads (Dynal) at a concentration of 8 μg of Fc-TALL-1 per 100 μl of the bead stock from the manufacturer. By drawing the beads to one side of a tube using a magnet and pipetting away the liquid, the beads were washed twice with the phosphate buffer saline (PBS) and resuspended in PBS. The Fc-TALL-1 protein was added to the washed beads at the above concentration and incubated with rotation for 1 hour at room temperature. The Fc-TALL-1 coated beads were then blocked by adding bovine serum albumin (BSA) to 1% final concentration and incubating overnight at 4° C. with rotation. The resulting Fc-TALL-1 coated beads were then washed twice with PBST (PBS with 0.05% Tween-20) before being subjected to the selection procedures.

[0366] B. Negative Selection Bead Preparation

[0367] Additional beads were also prepared for negative selections. For each panning condition, 250 μl of the bead stock from the manufacturer was subjected to the above procedure (section 1A) except that the incubation step with Fc-TALL-1 was omitted. In the last washing step, the beads were divided into five 50 μl aliquots.

[0368] 2. Selection of TALL-1 Binding Phage

[0369] A. Overall Strategy

[0370] Two filamentous phage libraries, TN8-IX (5×10⁹ independent transformants) and TN12-I (1.4×10⁹ independent transformants) (Dyax Corp.), were used to select for TALL-1 binding phage. Each library was subjected to either pH 2 elution or ‘bead elution’ (section 2E). Therefore, four different panning conditions were carried out for the TALL-1 project (TN8-IX using the pH2 elution method, TN8-IX using the bead elution method, TN12-I the using pH2 elution method, and TN12-I using the bead elution method). Three rounds of selection were performed for each condition.

[0371] B. Negative Selection

[0372] For each panning condition, about 100 random library equivalent (5×10¹¹ pfu for TN8-IX and 1.4×10¹¹ pfu for TN12-I) was aliquoted from the library stock and diluted to 300 μl with PBST. After the last washing liquid was drawn out from the first 50 μl aliquot of the beads prepared for negative selections (section 1B), the 300 μl diluted library stock was added to the beads. The resulting mixture was incubated for 10 minutes at room temperature with rotation. The phage supernatant was drawn out using the magnet and added to the second 50 μl aliquot for another negative selection step. In this way, five negative selection steps were performed.

[0373] C. Selection Using the Fc-TALL-1 Protein Coated Beads

[0374] The phage supernatant after the last negative selection step (section 1B) was added to the Fc-TALL-1 coated beads after the last washing step (section 1A). This mixture was incubated with rotation for two hours at room temperature, allowing specific phage to bind to the target protein. After the supernatant is discarded, the beads were washed seven times with PBST.

[0375] D. pH2 Elution of Bound Phage

[0376] After the last washing step (section 2C), the bound phages were eluted from the magnetic beads by adding 200 μl of CBST (50 mM sodium citrate, 150 mM sodium chloride, 0.05% Tween-20, pH2). After 5 minute incubation at room temperature, the liquid containing the eluted phage were drawn out and transferred to another tube. The elution step was repeated again by adding 200 μl of CBST and incubating for 5 minutes. The liquids from two elution steps were added together, and 100 μl of 2 M Tris solution (pH 8) was added to neutralize the pH. 500 μl of Min A Salts solution (60 mM K₂HPO₄, 33 mM KH₂PO₄, 7.6 mM (NH₄)SO₄, and 1.7 mM sodium citrate) was added to make the final volume to 1 ml.

[0377] E. ‘Bead Elution’

[0378] After the final washing liquid was drawn out (section 2C), 1 ml of Min A salts solution was added to the beads. This bead mixture was added directly to a concentrated bacteria sample for infection (section 3A and 3B).

[0379] 3. Amplification

[0380] A. Preparation of Plating Cells

[0381] Fresh E. Coli. (XL-1 Blue MRF′) culture was grown to OD₆₀₀=0.5 in LB media containing 12.5 μg/ml tetracycline. For each panning condition, 20 ml of this culture was chilled on ice and centrifuged. The bacteria pellet was resuspended in 1 ml of the Min A Salts solution.

[0382] B. Transduction

[0383] Each mixture from different elution methods (section 2D and 2E) was added to a concentrated bacteria sample (section 3A) and incubated at 37° C. for 15 minutes. 2 ml of NZCYM media (2XNZCYM, 50 μg/ml ampicillin) was added to each mixture and incubated at room temperature for 15 minutes. The resulting 4 ml solution was plated on a large NZCYM agar plate containing 50 μg/ml ampicillin and incubated overnight at 37° C.

[0384] C. Phage Harvesting

[0385] Each of the bacteria/phage mixture that was grown overnight on a large NZCYM agar plate (section 3B) was scraped off in 35 ml of LB media, and the agar plate was further rinsed with additional 35 ml of LB media. The resulting bacteria/phage mixture in LB media was centrifuged to pellet the bacteria away. 50 ml the of the phage supernatant was transferred to a fresh tube, and 12.5 ml of PEG solution (20% PEG8000, 3.5M ammonium acetate) was added and incubated on ice for 2 hours to precipitate phages. Precipitated phages were centrifuged down and resuspended in 6 ml of the phage resuspension buffer (250 mM NaCl, 100 mM Tris pH8, 1 mM EDTA). This phage solution was further purified by centrifuging away the remaining bacteria and precipitating the phage for the second time by adding 1.5 ml of the PEG solution. After a centrifugation step, the phage pellet was resuspended in 400 μl of PBS. This solution was subjected to a final centrifugation to rid of remaining bacteria debris. The resulting phage preparation was titered by a standard plaque formation assay (Molecular Cloning, Maniatis et al 3^(rd) Edition).

[0386] 4. Two More Rounds of Selection and Amplification

[0387] In the second round, the amplified phage (10¹⁰ pfu) from the first round (section 3C) was used as the input phage to perform the selection and amplification steps (sections 2 and 3). The amplified phage (10¹⁰ pfu) from the 2^(nd) round in turn was used as the input phage to perform 3^(rd) round of selection and amplification (sections 2 and 3). After the elution steps (sections 2D and 2E) of the 3^(rd) round, a small fraction of the eluted phage was plated out as in the plaque formation assay (section 3C). Individual plaques were picked and placed into 96 well microtiter plates containing 100 μl of TE buffer in each well. These master plates were incubated in a 37° C. incubator for 1 hour to allow phages to elute into the TE buffer.

[0388] 5. Clonal Analysis (Phage ELISA and Sequencing)

[0389] The phage clones were analyzed by phage ELISA and sequencing methods. The sequences were ranked based on the combined results from these two assays.

[0390] A. Phage ELISA

[0391] An XL-1 Blue MRF′ culture was grown until OD₆₀₀ reaches 0.5. 30 μl of this culture was aliquoted into each well of a 96 well microtiter plate. 10 μl of eluted phage (section 4) was added to each well and allowed to infect bacteria for 15 min at room temperature. 130 μl of LB media containing 12.5 μg/ml of tetracycline and 50 μg/ml of ampicillin was added to each well. The microtiter plate was then incubated overnight at 37° C. The recombinant TALL-1 protein (1 μg/ml in PBS) was allowed to coat onto the 96-well Maxisorp plates (NUNC) overnight and 4° C. As a control, the recombinant Fc-Trail protein was coated onto a separate Maxisorp plate at the same molar concentration as the TALL-1 protein. On the following day, liquids in the protein coated Maxisorp plates were discarded, and each well was blocked with 300 μl of 2% BSA solution at 37° C. for one hour. The BSA solution was discarded, and the wells were washed three times with the PBST solution. After the last washing step, 50 μl of PBST was added to each well of the protein coated Maxisorp plates. Each of the 50 μl overnight cultures in the 96 well microtiter plate was transferred to the corresponding wells of the TALL-1 coated plates as well as the control Fc-Trail coated plates. The 100 μl mixtures in the two kinds of plates were incubated for 1 hour at room temperature. The liquid was discarded from the Maxisorp plates, and the wells were washed five times with PBST. The HRP-conjugated anti-M13 antibody (Pharmacia) was diluted to 1:7,500, and 100 μl of the diluted solution was added to each well of the Maxisorp plates for 1 hour incubation at room temperature. The liquid was again discarded and the wells were washed seven times with PBST. 100 μl of tetramethylbenzidine (TMB) substrate (Sigma) was added to each well for the color reaction to develop, and the reaction was stopped with 50 μl of the 5 N H₂SO₄ solution. The OD₄₅₀ was read on a plate reader (Molecular Devices).

[0392] B. Sequencing of the Phage Clones.

[0393] For each phage clone, the sequencing template was prepared by a PCR method. The following oligonucleotide pair was used to amplify about 500 nucleotide fragment: (SEQ ID NO: 56) primer #1 (5′-CGGCGCAACTATCGGTATCAAGCTG-3′) (SEQ ID NO: 57) and primer #2 (5′-CATGTACCGTAACACTGAGTTTCGTC-3′).

[0394] The following mixture was prepared for each clone. Reagents volume (μL)/tube dH₂O 26.25 50% glycerol 10 10B PCR Buffer (w/o MgCl₂) 5 25 mM MgCl₂ 4 10 mM dNTP mix 1 100 μM primer 1 0.25 100 μM primer 2 0.25 Taq polymerase 0.25 Phage in TE (section 4) 3 Final reaction volume 50

[0395] The thermocycler (GeneAmp PCR System 9700, Applied Biosystems) was used to run the following program: 94° C. for 5 min; [94° C. for 30 sec, 55° C. for 30 sec, 72° C. for 45 sec.]×30 cycles; 72° C. for 7 min; cool to 4° C. The PCR product was checked by running 5 μl of each PCR reaction on a 1% agarose gel. The PCR product in the remaining 45 μl from each reaction was cleaned up using the QIAquick Multiwell PCR Purification kit (Qiagen), following the manufacturer's protocol. The resulting product was then sequenced using the ABI 377 Sequencer (Perkin-Elmer) following the manufacturer recommended protocol.

[0396] 6. Sequence Ranking and Consensus Sequence Determination

[0397] A. Sequence Ranking

[0398] The peptide sequences that were translated from variable nucleotide sequences (section 5B) were correlated to ELISA data. The clones that showed high OD₄₅₀ in the TALL-1 coated wells and low OD₄₅₀ in the Fc-Trail coated wells were considered more important. The sequences that occur multiple times were also considered important. Candidate sequences were chosen based on these criteria for further analysis as peptides or peptibodies. Five and nine candidate peptide sequences were selected from the TN8-IX and TN12-I libraries, respectively.

[0399] B. Consensus Sequence Determination

[0400] The majority of sequences selected from the TN12-I library contained a very conserved DBL motif. This motif was also observed in sequences selected from the TN8-IB library as well. Another motif, PFPWE (SEQ ID NO: 110) was also observed in sequences obtained from the TN8-IB library.

[0401] A consensus peptide, FHDCKWDLLTKQWVCHGL (SEQ ID NO: 58), was designed based on the DBL motif. Since peptides derived from the TN12-I library were the most active ones, the top 26 peptide sequences based on the above ranking criteria (section 5A) were aligned by the DBL motif. The underlined “core amino acid sequence” was obtained by determining the amino acid that occur the most in each position. The two cysteines adjacent to the core sequences were fixed amino acids in the TN12-I library. The rest of the amino acid sequence in the consensus peptide is taken from one of the candidate peptides, TALL-1-12-10 (Table 2, SEQ ID NO: 37). The peptide and peptibody that was derived from this consensus sequence were most active in the B cell proliferation assay.

EXAMPLE 2 Peptibodies

[0402] A set of 12 TALL-1 inhibitory peptibodies (Table 5) was constructed in which a monomer of each peptide was fused in-frame to the Fc region of human IgG1. Each TALL-1 inhibitory peptibody was constructed by annealing the pairs of oligonucleotides shown in Table 6 to generate a duplex encoding the peptide and a linker comprised of 5 glycine residues and one valine residue as an NdeI to SalI fragment. These duplex molecules were ligated into a vector (pAMG21-RANK-Fc, described herein) containing the human Fc gene, also digested with NdeI and SalI. The resulting ligation mixtures were transformed by electroporation into E. coli strain 2596 cells (GM221, described herein). Clones were screened for the ability to produce the recombinant protein product and to possess the gene fusion having the correct nucleotide sequence. A single such clone was selected for each of the peptibodies. The nucleotide and amino acid sequences of the fusion proteins are shown in FIGS. 4A through 4F. TABLE 5 Peptide sequences and oligonucleotides used to generate TALL-1 inhibitory peptibodies. Peptibody Antisense SEQ ID Sense oligo- Peptibody NO Peptide Sequence oligo-nucleotide nucleotide TALL-1-8-1-a 29 PGTCFPFPWECTHA 2517-24 2517-25 TALL-1-8-2-a 30 WGACWPFPWECFKE 2517-26 2517-27 TALL-1-8-4-a 31 VPFCDLLTKHCFEA 2517-28 2517-29 TALL-1-12-4-a 32 GSRCKYKWDVLTKQCFHH 2517-30 2517-31 TALL-1-12-3-a 33 LPGCKWDLLIKQWVCDPL 2517-32 2517-33 TALL-1-12-5-a 34 SADCYFDILTKSDVCTSS 2517-34 2517-35 TALL-1-12-8-a 35 SDDCMYDQLTRMFICSNL 2517-36 2517-37 TALL-1-12-9-a 36 DLNCKYDELTYKEWCQFN 2521-92 2521-93 TALL-1-12-10-a 37 FHDCKYDLLTRQMVCHGL 2521-94 2521-95 TALL-1-12-11-a 38 RNHCFWDHLLKQDICPSP 2521-96 2521-97 TALL-1-12-14-a 39 ANQCWWDSLTKKNVCEFF 2521-98 2521-99 TALL-1- 58 FHDCKWDLLTKQWVCHGL 2551-48 2551-49 consensus

[0403] TABLE 5B TALL-1 inhibitory peptibodies. Peptibody SEQ ID Peptibody NO Peptide Sequence TALL-1-8- 111 MPGTCFPFPW ECTHAGGGGG VDKTHTCPPC PAPELLGGPS 1-a VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK TALL-1-8- 112 MWGACWPFPW ECFKEGGGGG VDKTHTCPPC PAPELLGGPS 2-a VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKPNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK TALL-1-8- 113 MVPFCDLLTK HCFEAGGGGG VDKTHTCPPC PAPELLGGPS 4-a VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK TALL-1-12- 114 MGSRCKYKWD VLTKQCFHHG GGGGVDKTHT CPPCPAPELL 4-a GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK TALL-1-12- 115 MLPGCKWDLL IKQWVCDPLG GGGGVDKTHT CPPCPAPELL 3-a GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK TALL-1-12- 116 MSADCYFDIL TKSDVCTSSG GGGG VDKTHT CPPCPAPELL 5-a GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK TALL-1-12- 117 MSDDCMYDQL TRMFICSNLG GGGGVDKTHT CPPCPAPELL 8-a GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK TALL-1-12- 118 MDLNCKYDEL TYKEWCQFNG GGGGVDKTHT CPPCPAPELL 9-a GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK TALL-1-12- 119 MFHDCKYDLL TRQMVCHGLG GGGGVDKTHT CPPCPAPELL 10-a GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK TALL-1-12- 120 MRNHCFWDHL LKQDICPSPG GGGGVDKTHT CPPCPAPELL 11-a GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK TALL-1-12- 121 MANQCWWDSL TKKNVCEFFG GGGGVDKTHT CPPCPAPELL 14-a GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK TALL-1- 122 MFHDCKWDLL TKQWVCHGLG GGGGVDKTHT CPPCPAPELL consensus GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK TALL-1 12- 123 MLPGCKWDLL IKQWVCDPLG SGSATGGSGS TASSGSGSAT 3 tandem dimer HMLPGCKWDL LIKQWVCDPL GGGGGVDKTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS RDELTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK TALL-1 124 MFHDCKWDLL TKQWVCHGLG SGSATGGSGS TASSGSGSAT consensus tandem HMFHDCKWDL LTKQWVCHGL GGGGGVDKTH TCPPCPAPEL dimer LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS RDELTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK

[0404] TABLE 6 Sequences of oligonucleotides used in peptibody construction. Oligo- nucleotide ID SEQ number ID NO Sequence 2517-24 71 TAT GCC GGG TAC TTG TTT CCC GTT CCC GTG GGA ATG CAC TCA CGC TGG TGG AGG CGG TGG GG 2517-25 72 TCG ACC CCA CCG CCT CCT GGA GCG TGA GTG CAT TCC CAC GGG AAG CCG AAA CAA GTA CCC GGC A 2517-26 73 TAT GTG GGG TGC TTG TTG GCC GTT CCC GTG GGA ATG TTT CAA AGA AGG TGG AGG CGG TGG GG 2517-27 74 TCG ACC CCA CCG CCT CCA CCT TCT TTG AAA CAT TCC CACGGG AAC GGC CAA CAAGCA CCC CAC A 2517-28 75 TAT GGT TCC GTT CTG TGA CCT GCT GAC TAA ACA CTG TTT CGA AGC TGG TGG AGG CGG TGG GG 2517-29 76 TCG ACC CCA CCG CCT CCA CCA GCT TCG AAA CAG TGT TTA GTC AGC AGG TCA CAGAAC GGA ACC A 2517-30 77 TAT GGG TTC TCG TTG TAA ATA CAA ATG GGA CGT TCT GAC TAA ACA GTG TTT CCA CCA CGG TGG AGG CGG TGG GG 2517-31 78 TCG ACC CCA CCG CCT CCA CCG TGG TGG AAA CAC TGT TTA GTC AGA ACG TCC CAT TTG TAT TTA CAA CGA GAA CCC A 2517-32 79 TAT GCT GCC GGG TTG TAA ATG GGA CCT GCT GAT CAA ACA GTG GGT TTG TGA CCC GCT GGG TGG AGG CGG TGG GG 2517-33 80 TCG ACC CCA CCG CCT CCA CCC AGC GGG TCA CAA ACC CAC TGT TTG ATC AGC AGG TCC CAT TTA CAA CCC GGC AGC A 2517-34 81 TAT GTC TGC TGA CTG TTA CTT CGA CAT CCT GAC TAA ATC TGA CGT TTG TAC TTC TTC TGG TGG AGG CGG TGG GG 2517-35 82 TCG ACC CCA CCG CCT CCA CCA GAA GAA GTA CAA ACG TCA GAT TTA GTC AGG ATG TCG AAG TAA CAG TCA GCA GAC A 2517-36 83 TAT GTC TGA CGA CTG TAT GTA CGA CCA GCT GAC TCG TAT GTT CAT CTG TTC TAA CCT GGG TGG AGG CGG TGG GG 2517-37 84 TCG ACC CCA CCG CCT CCA CCC AGG TTA GAA CAG ATG AAC ATA CGA GTC AGC TGG TCG TAC ATA CAG TCG TCA GAC A 2521-92 85 TAT GGA CCT GAA CTG TAA ATA CGA CGA ACT GAC TTA CAA AGA ATG GTG TCA GTT CAA CGG TGG AGG CGG TGG GG 25221-93 86 TCG ACC CCA CCG CCT CCA CCG TTG AAC TGA CAC CAT TCT TTG TAA GTC AGTTCG TCG TAT TTA CAG TTC AGG TCC A 2521-94 87 TAT GTT CCA CGA CTG TAA ATA CGA CCT GCT GAC TCG TCA GAT GGT TTG TCA CGG TCT GGG TGG AGG CGG TGG GG 2521-95 88 TCG ACC CCA CCG CCT CCA CCC AGA CCG TGA CAA ACC ATC TGA CGA GTC AGC AGG TCG TAT TTA CAG TCG TGG AAC A 2521-96 89 TAT GCG TAA CCA CTG TTT CTG GGA CCA CCT GCT GAA ACA GGA CAT CTG TCC GTC TCC GGG TGG AGG CGG TGG GG 2521-97 90 TCG ACC CCA CCG CCT CCA CCC GGA GAC GGA CAG ATG TCC TGT TTC AGC AGG TGG TCC CAG AAA CAG TGG TTA CGC A 2521-98 91 TAT GGC TAA CCA GTG TTG GTG GGA CTC TCT GCT GAA AAA AAA CGT TTG TGA ATT CTT CGG TGG AGG CGG TGG GG 2521-99 92 TCG ACC CCA CCG CCT CCA CCG AAG AAT TCA CAA ACG TTT TTT TTC AGC AGA GAG TCC CAC CAA CAC TGG TTA GCC A 2551-48 93 TAT GTT CCA CGA CTG CAA ATG GGA CCT GCT GAC CAA ACA GTG GGT TTG CCA CGG TCT GGG TGG AGG CGG TGG GG 2551-49 94 TCG ACC CCA CCG CCT CCA CCC AGA CCG TGG CAA ACC CAC TGT TTG GTC AGC AGG TCC CAT TTG CAG TCG TGG AAC A

[0405] pAMG21-RANK-Fc Vector

[0406] pAMG21. The expression plasmid pAMG21 (ATCC accession no. 98113) can be derived from the Amgen expression vector pCFM1656 (ATCC #69576) which in turn be derived from the Amgen expression vector system described in U.S. Pat. No. 4,710,473. The pCFM1656 plasmid can be derived from the described pCFM836 plasmid (U.S. Pat. No. 4,710,473) by:

[0407] destroying the two endogenous NdeI restriction sites by end filling with T4 polymerase enzyme followed by blunt end ligation;

[0408] replacing the DNA sequence between the unique AatII and ClaI restriction sites containing the synthetic P_(L) promoter with a similar fragment obtained from pCFM636 (U.S. Pat. No. 4,710,473) containing the P_(L) promoter (see SEQ ID NO: 95 below); and

[0409] substituting the small DNA sequence between the unique ClaI and KpnI restriction sites with the oligonucleotide having the sequence of SEQ ID NO: 96. SEQ ID NO: 95: AatII 5′  CTAATTCCGCTCTCACCTACCAAACAATGCCCCCCTGCAAAAAATAAATTCATAT- 3′  TGCAGATTAAGGCGAGAGTGGATGGTTTGTTACGGGGGGACGTTTTTTATTTAAGTATA-     -AAAAAACATACAGATAACCATCTGCGGTGATAAATTATCTCTGGCGGTGTTGACATAAA-     -TTTTTTGTATGTCTATTGGTAGACGCCACTATTTAATAGAGACCGCCACAACTGTATTT-     -TACCACTGGCGGTGATACTGAGCACAT 3′     -ATGGTGACCGCCACTATGACTCGTGTAGC 5′                                ClaI SEQ ID NO: 96: 5′  CGATTTGATTCTAGAAGGAGGAATAACATATGGTTAACGCGTTGGAATTCGGTAC 3′ 3′  TAAACTAAGATCTTCCTCCTTATTGTATACCAATTGCGCAACCTTAAGC 5′    ClaI                                           KpnI

[0410] The expression plasmid pAMG21 can then be derived from pCFM1656 by making a series of site-directed base changes by PCR overlapping oligonucleotide mutagenesis and DNA sequence substitutions. Starting with the BglII site (plasmid bp # 180) immediately 5′ to the plasmid replication promoter P_(copB) and proceeding toward the plasmid replication genes, the base pair changes are as shown in Table 7 below. +M1 TABLE 7+HZ,1/32 !+HC,1 +UZ,5/28 Base pair changes resulting in pAMG21? +HL,32 ? !+HL,1 pAMG21 bp #? +HL,8 bp in pCFM1656? +HL,16 bp changed to in pAMG21+HZ,1/32 ? !,1 #204? ,8 T/A? ,16 C/G? ,32 ? ! !#428? A/T? G/C? ! !#509? G/C? A/T? ! !#617? --? insert two G/C bp? ! !#679? G/C? T/A? ! !#980? T/A? C/G? ! !#994? G/C? A/T? ! !#1004? A/T? C/G? ! !#1007? C/G? T/A? ! !#1028? A/T? T/A? ! !#1047? C/G? T/A? ! !#1178? G/C? T/A? ! !#1466? G/C? T/A? ! !#2028? G/C? bp deletion? ! !#2187? C/G? T/A? ! !#2480? A/T? T/A? ! !#2499-2502? AGTG? GTCA? !? TCAC? CAGT? ! !#2642? TCCGAGC? 7 bp deletion? !? AGGCTCG? ! !#3435? G/C? A/T? ! !#3446? G/C? A/T? ! !#3643? A/T? T/A+TZ,1/32 ? !!

[0411] The DNA sequence between the unique AatII (position #4364 in pCFM1656) and SacII (position #4585 in pCFM1656) restriction sites is substituted with the DNA sequence below (SEQ ID NO: 97):.

0 [AatII sticky end] 5′ GCGTAACGTATGCATGGTCTCC- (position #4358 in pAMG21) 3′TGCACGCATTGCATACGTACCAGAGG- -CCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACT- -GGTACGCTCTCATCCCTTGACGGTCCGTAGTTTATTTTGCTTTCCGAGTCAGCTTTCTGA- -GGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGC- -CCCGGAAAGCAAAATAGACAACAAACAGCCACTTGCGAGAGGACTCATCCTCTTTAGGCG- -CGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGC- -GCCCTCGCCTAAACTTGCAACGCTTCGTTGCCGGGCCTCCCACCGCCCGTCCTGCGGGCG- -CATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGT- -GTATTTGACGGTCCGTAGTTTAATTCGTCTTCCGGTAGGACTGCCTACCGGAAAAACGCA- AatII -TTCTACAAACTCTTTTGTTTATTTTTCTAAATACATTCAAATATGGACGTCGTACTTAAC- -AAGATGTTTGAGAAAACAAATAAAAAGATTTATGTAAGTTTATACCTGCAGCATGAATTG- -TTTTAAAGTATGGGCAATCAATTGCTCCTGTTAAAATTGCTTTAGAAATACTTTGGCAGC- -AAAATTTCATACCCGTTAGTTAACGAGGACAATTTTAACGAAATCTTTATGAAACCGTCG- -GGTTTGTTGTATTGAGTTTCATTTGCGCATTGGTTAAATGGAAAGTGACCGTGCGCTTAC- -CCAAACAACATAACTCAAAGTAAACGCGTAACCAATTTACCTTTCACTGGCACGCGAATG- -TACAGCCTAATATTTTTGAAATATCCCAAGAGCTTTTTCCTTCGCATGCCCACGCTAAAC- -ATGTCGGATTATAAAAACTTTATAGGGTTCTCGAAAAAGGAAGCGTACGGGTGCGATTTG- -ATTCTTTTTCTCTTTTGGTTAAATCGTTGTTTGATTTATTATTTGCTATATTTATTTTTC- -TAAGAAAAAGAGAAAACCAATTTAGCAACAAACTAAATAATAAACGATATAAATAAAAAG- -GATAATTATCAACTAGAGAAGGAACAATTAATGGTATGTTCATACACGCATGTAAAAATA- -CTATTAATAGTTGATCTCTTCCTTGTTAATTACCATACAAGTATGTGCGTACATTTTTAT- -AACTATCTATATAGTTGTCTTTCTCTGAATGTGCAAAACTAAGCATTCCGAAGCCATTAT- -TTGATAGATATATCAACAGAAAGAGACTTACACGTTTTGATTCGTAAGGCTTCGGTAATA- -TAGCAGTATGAATAGGGAAACTAAACCCAGTGATAAGACCTGATGATTTCGCTTCTTTAA- -ATCGTCATACTTATCCCTTTGATTTGGGTCACTATTCTGGACTACTAAAGCGAAGAAATT- -TTACATTTGGAGATTTTTTATTTACAGCATTGTTTTCAAATATATTCCAATTAATCGGTG- -AATGTAAACCTCTAAAAAATAAATGTCGTAACAAAAGTTTATATAAGGTTAATTAGCCAC- -AATGATTGGAGTTAGAATAATCTACTATAGGATCATATTTTATTAAATTAGCGTCATCAT- -TTACTAACCTCAATCTTATTAGATGATATCCTAGTATAAAATAATTTAATCGCAGTAGTA- -AATATTGCCTCCATTTTTTAGGGTAATTATCCAGAATTGAAATATCAGATTTAACCATAG- -TTATAACGGAGGTAAAAAATCCCATTAATAGGTCTTAACTTTATAGTCTAAATTGGTATC- -AATGAGGATAAATGATCGCGAGTAAATAATATTCACAATGTACCATTTTAGTCATATCAG- -TTACTCCTATTTACTAGCGCTCATTTATTATAAGTGTTACATGGTAAAATCAGTATAGTC- -ATAAGCATTGATTAATATCATTATTGCTTCTACAGGCTTTAATTTTATTAATTATTCTGT- -TATTCGTAACTAATTATAGTAATAACGAAGATGTCCGAAATTAAAATAATTAATAAGACA- -AAGTGTCGTCGGCATTTATGTCTTTCATACCCATCTCTTTATCCTTACCTATTGTTTGTC- -TTCACAGCAGCCGTAAATACAGAAAGTATGGGTAGAGAAATAGGAATGGATAACAAACAG- -GCAAGTTTTGCGTGTTATATATCATTAAAACGGTAATAGATTGACATTTGATTCTAATAA- -CGTTCAAAACGCACAATATATAGTAATTTTGCCATTATCTAACTGTAAACTAAGATTATT- -ATTGGATTTTTGTCACACTATTATATCGCTTGAAATACAATTGTTTAACATAAGTACCTG- -TAACCTAAAAACAGTGTGATAATATAGCGAACTTTATGTTAACAAATTGTATTCATGGAC- -TAGGATCGTACAGGTTTACGCAAGAAAATGGTTTGTTATAGTCGATTAATCGATTTGATT- -ATCCTAGCATGTCCAAATGCGTTCTTTTACCAAACAATATCAGCTAATTAGCTAAACTAA- -CTAGATTTGTTTTAACTAATTAAAGGAGGAATAACATATGGTTAACGCGTTGGAATTCGA- -GATCTAAACAAAATTGATTAATTTCCTCCTTATTGTATACCAATTGCGCAACCTTAAGCT- SacII -GCTCACTAGTGTCGACCTGCAGGGTACCATGGAAGCTTACTCGAGGATCCGCGGAAAGAA- -CGAGTGATCACAGCTGGACGTCCCATGGTACCTTCGAATGAGCTCCTAGGCGCCTTTCTT- -GAAGAAGAAGAAGAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATA- -CTTCTTCTTCTTCTTTCGGGCTTTCCTTCGACTCAACCGACGACGGTGGCGACTCGTTAT- -ACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGG- -TGATCGTATTGGGGAACCCCGGAGATTTGCCCAGAACTCCCCAAAAAACGACTTTCCTCC- -AACCGCTCTTCACGCTCTTCACGC 3′ [SacII sticky end] -TTGGCGAGAAGTGCGAGAAGTG 5′ (position #5904 in pAMG21) 

What is claimed is:
 1. A TALL-1-binding composition of matter comprising an amino acid sequence Dz²Lz⁴, wherein z² is an amino acid residue and z⁴ is T or I, and wherein the composition of matter does not comprise a fragment of TACI, BCMA, or BAFFR (SEQ ID NOS: 195, 196, and 197).
 2. The composition of matter of claim 1, wherein z⁴ is T.
 3. A TALL-1-binding composition of matter comprising an amino acid sequence Dz²LI, wherein z² is an amino acid residue.
 4. The composition of matter of claim 1 comprising an amino acid sequence of the formula +M1 !,1 a¹a²a³CDa⁶La⁸a⁹a¹⁰Ca¹²a¹³a¹⁴? ,20 (SEQ. ID. NO: 100)? ,32 ? ! ! wherein: a¹, a², a³ are each independently absent or amino acid residues; a⁶ is an amino acid residue; a⁸is T or I; a⁹ is a basic or hydrophobic residue; a¹² is a neutral polar residue; and a¹³ and a¹⁴ are each independently absent or amino acid residues.
 5. The composition of matter of claim 4 wherein a⁸ is T and a⁹ is a basic residue.
 6. The composition of matter of claim 4 wherein a⁹ is K and a¹² is F.
 7. The composition of matter of claim 1 comprising an amino acid sequence of the formula +M1 !+TR,4 (SEQ. ID. NO: 104)?,29 ? ,32 ? !,4 b¹b²b³Cb⁵b⁶Db⁸Lb¹⁰b¹¹b¹²b¹³b¹⁴Cb¹⁶b¹⁷b¹⁸? ,32 ? ! ! wherein: b¹ and b² are each independently absent or amino acid residues; b³ is an acidic or amide residue; b⁵ is an amino acid residue; b⁶ is an aromatic residue; b⁸ is an amino acid residue; b¹⁰ is T or I; b¹¹ is a basic residue; b¹² and b¹³ are each independently amino acid residues; b¹⁴ is a neutral polar residue; and b¹⁶, b¹⁷, and b¹⁸ are each independently absent or amino acid residues.
 8. The composition of matter of claim 7 wherein: b³ is D, Q, or E; b⁶ is W or Y; b¹⁰ is T; b¹¹ is K or R; and b¹⁴is V or L.
 9. The composition of matter of claim 1 comprising an amino acid sequence of the formula +M1 !+TR,4 (SEQ. ID. NO: 105)?,29 ? ,32 ? !,4 c¹c²c³Cc⁵Dc⁷Lc⁹c¹⁰c¹¹c¹²c¹³c¹⁴Cc¹⁶c¹⁷c¹⁸? ,32 ? ! ! wherein: c¹, c², and c³ are each independently absent or amino acid residues; c⁵ is an amino acid residue; c⁷ is an amino acid residue; c⁹ is T or I; c¹⁰ is a basic residue; c¹¹ and c¹² are each independently amino acid residues; c¹³ is a neutral polar residue; c¹⁴ is an amino acid residue; c¹⁶ is an amino acid residue; c¹⁷ is a neutral polar residue; and c¹⁸ is an amino acid residue or is absent.
 10. The composition of matter of claim 9 wherein: c⁹ is T; c¹⁰ is K or R; c¹³ is a I, L, or V; and c¹⁷ is A or L.
 11. The composition of matter of claim 1 comprising an amino acid sequence of the formula +M1 !+TR,1 (SEQ. ID. NO: 106)? ,28 ? ,32 ? !,1 d¹d²d³Cd⁵d⁶d⁷WDd¹⁰Ld¹²d¹³d¹⁴Cd¹⁵d¹⁶d¹⁷? ,32 ? ! ! wherein: d¹, d², and d³ are each independently absent or amino acid residues; d⁵, d⁶, and d⁷ are each independently amino acid residues; d¹⁰ is an amino acid residue; d¹³ is T or I; d¹⁴ is an amino acid residue; and d¹⁶, d¹⁷, and d¹⁸ are each independently absent or amino acid residues.
 12. The composition of matter of claim 1 comprising an amino acid sequence of the formula +M1 !+TR,1 (SEQ. ID. NO: 107)? ,28 ? ,32 ? !,1 e¹e²e³Ce⁵e⁶e⁷De⁹L e¹¹Ke¹³Ce¹⁵e¹⁶e¹⁷e¹⁸? ,32 ? ! ! wherein: e¹, e², and e³ are each independently absent or amino acid residues; e⁵, e⁶, e⁷, e⁹, and e¹³ are each independently amino acid residues; e¹¹ is T or I; and e¹⁵, e¹⁶, and e¹⁷ are each independently absent or amino acid residues.
 13. The composition of matter of claim 1 comprising an amino acid sequence of the formula +M1 !,1 f¹f²f³Kf⁵Df⁷Lf⁹f¹⁰Qf¹²f¹³f¹⁴? ,21 (SEQ ID NO: 109)? ,32 ? ! ! wherein: f¹, f², and f³ are absent or are amino acid residues; f⁵ is W, Y, or F; f⁷ is an amino acid residue; f⁹ is T or I; f¹⁰ is K, R, or H; f¹² is C, a neutral polar residue, or a basic residue (W, C, or R preferred); f¹³ is C, a neutral polar residue or is absent; and f¹⁴ is any amino acid residue or is absent; provided that only one of f¹, f², and f³ may be C, and only one of f¹², f¹³, and f¹⁴ may be C.
 14. The composition of matter of claim 13, wherein f⁵ is W.
 15. The composition of matter of claim 13, wherein f⁷ is L.
 16. The composition of matter of claim 13, wherein f⁹ is T.
 17. The composition of matter of claim 13, wherein f¹⁰ is K.
 18. The composition of matter of claim 13, wherein f¹² is C and one of f¹, f², and f³ is C.
 19. The composition of matter of claim 13, wherein f¹³ is V.
 20. The composition of matter of claim 13 comprising an amino acid sequence of the formula +M1 !,3 f¹f²f³KWDf⁷Lf⁹KQf¹²f¹³f^(14.)? ,20 (SEQ ID NO: 125)? ,32 ? ! !
 21. The composition of matter of claim 20 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 32, 33, 58, 60, 63, 66, 67, 69, 114, 115, 122, 123, 124, 147-150, 152-177, 179, 180, and
 187. 22. The composition of matter of claim 20 comprising an amino acid sequence of the formula LPGCKWDLLIKQWVCDPL (SEQ ID NO: 33).
 23. A composition of matter comprising an amino acid sequence of the formula +M1 !,3 g¹g²g³Cg⁵PFg⁸Wg¹⁰Cg¹¹g¹²g¹³? ,21 (SEQ. ID. NO: 101)? ,32 ? ! ! wherein: g¹, g² and g³ are each independently absent or amino acid residues; g⁵ is a neutral polar residue; g⁸ is a neutral polar residue; g¹⁰ is an acidic residue; g¹² and g¹³ are each independently amino acid residues; and g¹⁴ is absent or is an amino acid residue.
 24. The composition of matter of claim 23 wherein: g² is G; g⁵ is W; g⁸ is P; g¹⁰ is E; and g¹³ is a basic residue.
 25. A composition of matter comprising an amino acid sequence of the formula +M1 !,3 h¹h²h³CWh⁶h⁷WGh¹⁰Ch¹²h¹³h¹⁴? ,21 (SEQ. ID. NO: 102)? ,32 ? ! ! wherein: h¹, h², and h³ are each independently absent or amino acid residues; h⁶ is a hydrophobic residue; h⁷ is a hydrophobic residue; h¹⁰ is an acidic or polar hydrophobic residue; and h¹², h¹³, and h¹⁴ are each independently absent or amino acid residues.
 26. The composition of matter of claim 25 wherein: h¹ is G; h⁶ is A; h⁷ is a neutral polar residue; and h¹⁰ is an acidic residue.
 27. A composition of matter comprising an amino acid sequence of the formula +M1 !,1 i¹i²i³Ci⁵i⁶i⁷i⁸i⁹i¹⁰Ci¹²i¹³i¹⁴? ,21 (SEQ. ID. NO: 103)? ,32 ? ! ! wherein: i¹ is absent or is an amino acid residue; i² is a neutral polar residue; i³is an amino acid residue; i⁵, i⁶, i⁷, and i⁸ are each independently amino acid residues; i⁹ is an acidic residue; i¹⁰ is an amino acid residue; i¹² and i¹³ are each independently amino acid residues; and i¹⁴ is a neutral polar residue.
 28. The composition of matter of claim 27 wherein: i² is W; and i¹⁴ is W.
 29. A TALL-1 binding composition of matter comprising an amino acid sequence of the formula PFPWE (SEQ ID NO: 110).
 30. The composition of matter of claim 1 having the formula (X¹)_(a)—V¹—(X²)_(b) and multimers thereof, wherein: V¹ is a vehicle; X¹ and X² are each independently selected from —(L¹)_(c)—P¹, —(L¹)_(c)—P¹—(L²)_(d)—P², —(L¹)_(c)—P¹—(L²)_(d)—P²—(L³)₃—P³, and —(L¹)_(c)—P¹—(L²)_(d)—P²—(L³)_(e)—P³—(L⁴)_(f)—P^(4 one or more of P) ¹, P², P³, and P⁴ each independently comprise Dz²Lz⁴; L¹, L², L³, and L⁴ are each independently linkers; and a, b, c, d, e, and f are each independently 0 or 1, provided that at least one of a and b is
 1. 31. The composition of matter of claim 30 of the formula P¹—(L¹)_(c)—P²—(L²)_(d).—V¹.
 32. The composition of matter of claim 30 of the formula V¹—(L¹)_(c)—P¹—(L²)_(d)—P².
 33. The composition of matter of claim 30, wherein V¹ is an Fc domain.
 34. The composition of matter of claim 30 wherein V¹ is an IgG Fc domain.
 35. The composition of matter of claim 30 wherein V¹ is an IgG1 Fc domain.
 36. The composition of matter of claim 30 wherein V¹ comprises the sequence of SEQ ID NO:
 2. 37. The composition of matter of claim 30 wherein one or more of P¹, P², P³, and P⁴ each independently comprises a sequence selected from: +M1 !+TR,1 (SEQ. ID. NO: 100)? ,29 ? ,32 ? !,4 a¹a²a³CDa⁶La⁸a⁹a¹⁰Ca¹²a¹³a¹⁴? ,32 ? ! !+TR,1 (SEQ. ID. NO: 104)? ,29 ? ,32 ? !,4 b¹b²b³Cb⁵b⁶Db⁸Lb¹⁰b¹¹b¹²b¹³b¹⁴Cb¹⁶b¹⁷b¹⁸? ,32 ? ! !+TR,1 (SEQ. ID .NO: 105)? ,29 ? ,32 ? !,4 c¹c²c³Cc⁵Dc⁷Lc⁹c¹⁰c¹¹c¹²c¹³c¹⁴Cc¹⁶c¹⁷c¹⁸? ,32 ? ! !+TR,1 (SEQ. ID. NO: 106)? ,29 ? ,32 ? !,4 d¹d²d³Cd⁵d⁶d⁷WDd¹⁰Ld¹³d¹⁴d¹⁵Cd¹⁶d¹⁷d¹⁸? ,32 ? ! !+TR,1 (SEQ. ID. NO: 107)? ,29 ? ,32 ? !,4 e¹e²e³Ce⁵e⁶e⁷De⁹Le¹¹Ke¹³Ce¹⁵e¹⁶e¹⁷e¹⁸? ,32 ? ! !+TR,1 (SEQ. ID. NO: 109)? ,29 ? ,32 ? !,4 f¹f²f³Kf⁵Df⁷Lf⁹f¹⁰Qf¹²f¹³f¹⁴? ,32 ? ! !+TR,1 (SEQ ID NO: 101)? ,29 ? ,32 ? !,4 g¹g²g³Cg⁵PFg⁸Wg¹⁰Cg¹¹g¹²g¹³,? ,32 ? ! !+TR,1 (SEQ ID NO: 102)? ,29 ? ,32 ? !,4 h¹h²h³CWh⁶h⁷WGh¹⁰Ch¹²h¹³h¹⁴, and? ,32 ? ! !+TR,1 (SEQ ID NO: 103)? ,29 ? ,32 ? !,4 i¹i²i³Ci⁵i⁶i⁷i⁸i⁹i¹⁰Ci¹²i¹i¹⁴? ,32 ? ! ! wherein: a¹, a², a³ are each independently absent or amino acid residues; a⁶ is an amino acid residue; a⁹ is a basic or hydrophobic residue; a⁸ is threonyl or isoleucyl; a¹² is a neutral polar residue; a¹³ and a¹⁴ are each independently absent or amino acid residues; b¹ and b² are each independently absent or amino acid residues; b³ is an acidic or amide residue; b⁵ is an amino acid residue; b⁶ is an aromatic residue; b⁸ is an amino acid residue; b¹⁰ is T or I; b¹¹ is a basic residue; b¹² and b¹³ are each independently amino acid residues; b¹⁴ is a neutral polar residue; b¹⁶, b¹⁷, and b¹⁸ are each independently absent or amino acid residues; c¹, c², and c³ are each independently absent or amino acid residues; c⁵ is an amino acid residue; c⁷is an amino acid residue; c⁹is T or I; c¹⁰ is a basic residue; c¹¹ and c¹² are each independently amino acid residues; c¹³ is a neutral polar residue; c¹⁴ is an amino acid residue; c¹⁶ is an amino acid residue; c¹⁷ is a neutral polar residue; and c¹⁸ is an amino acid residue or is absent; d¹, d², and d³ are each independently absent or amino acid residues; d⁵, d⁶, and d⁷ are each independently amino acid residues; d¹⁰ is an amino acid residue; d¹² is T or I; d¹³ is an amino acid residue; and d¹⁵, d¹⁶, and d¹⁷ are each independently absent or amino acid residues; e¹, e², and e³ are each independently absent or amino acid residues; e⁵, e⁶, e⁷, e⁹, and e¹³ are each independently amino acid residues; e¹¹ is T or I; and e¹⁵, e₁₆, and e¹⁷ are each independently absent or amino acid residues; f¹, f², and f³ are absent or are amino acid residues; f⁵ is W, Y, or F; f⁷is an amino acid residue; f⁹ is T or I; f¹⁰ is K, R, or H; f¹² is C, a neutral polar residue, or a basic residue; f¹³ is C, a neutral polar residue or is absent; and f¹⁴ is any amino acid residue or is absent; provided that only one of f¹, f², and f³ may be C, and only one of f¹², f¹³, and f¹⁴ may be C; g¹, g², and g³ are each independently absent or amino acid residues; g⁵ is a neutral polar residue; g⁸ is a neutral polar residue; g¹⁰ is an acidic residue; g¹² and g¹³ are each independently amino acid residues; and g¹⁴ is absent or is an amino acid residue; h¹, h², and h³ are each independently absent or amino acid residues; h⁶ is a hydrophobic residue; h⁷ is a hydrophobic residue; h¹⁰ is an acidic or polar hydrophobic residue; and h¹², h¹³, and h¹⁴ are each independently absent or amino acid residues; i¹ is absent or is an amino acid residue; i² is a neutral polar residue; i³ is an amino acid residue; i⁵, i⁶, i⁷, and i⁸ are each independently amino acid residues; i⁹ is an acidic residue; i¹⁰ is an amino acid residue; i¹² and i¹³ are each independently amino acid residues; and i¹⁴ is a neutral polar residue.
 38. The composition of matter of claim 37, wherein: a⁹ is a basic residue. b³ is D, Q, or E; b⁶ is W or Y; b¹¹ is K or R; and b¹⁴ is V or L. c¹⁰ is K or R; c¹³ is a I, L, or V; c¹⁷ is A or L; f⁵ is W; f⁷ is L; f⁷ is K; and f¹⁰ is V.
 39. The composition of matter of claim 37, wherein one or more of P¹, P², P³, and P⁴ each independently comprises +M1 !,3 f¹f²f³KWDf⁷Lf⁹KQf¹²f¹³f¹⁴.? ,20 (SEQ ID NO: 125)? ,32 ? ! !
 40. The composition of matter of claim 39 of the formula P¹—(L¹)_(c)—P²—(L²)_(d).—V¹.
 41. The composition of matter of claim 39 of the formula V¹—(L¹)_(c)—P¹—(L²)_(d)—P².
 42. The composition of matter of claim 39 having an amino acid sequence selected from SEQ ID NOS: 122, 123, and
 124. 43. The composition of matter of claim 40 wherein L² is greater than 5 amino acids.
 44. The composition of matter of claim 43 wherein L² is selected from +M1 !+TR,1 (SEQ ID NO: 193)? ,32 ? !,1 GSGSATGGSGSTASSGSGSATx¹x² and? ,32 ? ! !+TR,1 (SEQ ID NO: 194)? ,32 ? !,1 GSGSATGGSGSTASSGSGSATx¹x²GSGSATGGSGSTASSGSGSATx³x⁴? ,32 ? ! ! wherein x¹ and x³ are each independently basic or hydrophobic residues and x² and x⁴ are each independently hydrophobic residues.
 45. The composition of matter of claim 41 wherein L² is selected from +M1 !+TR,3 (SEQ ID NO: 59)? ,32 ? !,3 GSGSATGGSGSTASSGSGSATH,? ,32 ? ! !+TR,3 (SEQ ID NO: 190)? ,32 ? !,3 GSGSATGGSGSTASSGSGSATGM? ,32 ? ! !+TR,3 (SEQ ID NO: 191)?,32 ? !,3 GSGSATGGSGSTASSGSGSATGS,? ,32 ? !and? ! !+TR,3 (SEQ ID NO: 192)? ,32 ? !,3 GSGSATGGSGSTASSGSGSATHMGSGSATGGSGSTASSGSGSATHM.? ,32 ? ! !
 46. The composition of matter of claim 28 comprising a sequence selected from Table 2 (SEQ ID NOS: 29-39, 60-70, and 126-188).
 47. The composition of matter of claim 30 comprising a sequence selected from Table 4 (SEQ ID NOS: 44-55).
 48. The composition of matter of claim 46, wherein V¹ is an Fc domain.
 49. The composition of matter of claim 46, wherein V¹ is an IgG Fc domain.
 50. The composition of matter of claim 46, wherein V¹ is an IgG1 Fc domain.
 51. A DNA encoding a composition of matter of claim
 34. 52. An expression vector comprising the DNA of claim
 51. 53. A host cell comprising the expression vector of claim
 52. 54. The cell of claim 53, wherein the cell is an E. coli cell.
 55. A method of treating a B-cell mediated autoimmune disease, which comprises administering a composition of matter of claim
 1. 56. A method of treating a B-cell mediated autoimmune disease, which comprises administering a composition of matter of claim
 13. 57. A method of treating lupus, which comprises administering a composition of matter of claim
 1. 58. A method of treating lupus, which comprises administering a composition of matter of claim
 13. 59. A method of treating a B-cell mediated cancer, which comprises administering a composition of matter of claim
 1. 60. A method of treating a B-cell mediated cancer, which comprises administering a composition of matter of claim
 13. 61. A method of treating B-cell lymphoma, which comprises administering a composition of matter of claim
 1. 62. A method of treating B-cell lymphoma, which comprises administering a composition of matter of claim
 13. 